U.S. patent application number 11/103940 was filed with the patent office on 2005-11-17 for fra-1 expression in brain cancer.
This patent application is currently assigned to The Penn State Research Foundation. Invention is credited to Debinski, Waldemar, Gibo, Denise M..
Application Number | 20050255510 11/103940 |
Document ID | / |
Family ID | 23021421 |
Filed Date | 2005-11-17 |
United States Patent
Application |
20050255510 |
Kind Code |
A1 |
Debinski, Waldemar ; et
al. |
November 17, 2005 |
Fra-1 expression in brain cancer
Abstract
Fra-1 serves as a target for diagnosing and treating
glioblastoma multiforme and related brain cancers. Cancer in a
brain tissue sample is detected by analyzing expression of Fra-1 in
the sample. Brain cancer is treated by modulating Fra-1 gene
expression in cells of the cancer, and by inhibiting angiogenesis
associated with the cancer by interfering with Fra-1 binding to a
VEGF-D promoter.
Inventors: |
Debinski, Waldemar;
(Winston-Salem, NC) ; Gibo, Denise M.;
(Winston-Salem, NC) |
Correspondence
Address: |
AKERMAN SENTERFITT
P.O. BOX 3188
WEST PALM BEACH
FL
33402-3188
US
|
Assignee: |
The Penn State Research
Foundation
University Park
PA
|
Family ID: |
23021421 |
Appl. No.: |
11/103940 |
Filed: |
April 12, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11103940 |
Apr 12, 2005 |
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10075499 |
Feb 12, 2002 |
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6884581 |
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60268089 |
Feb 12, 2001 |
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Current U.S.
Class: |
435/6.18 ;
435/91.2 |
Current CPC
Class: |
C12Q 1/6886 20130101;
G01N 33/57484 20130101; G01N 33/5011 20130101; A61K 38/00 20130101;
C12Q 2600/158 20130101; C07K 14/82 20130101 |
Class at
Publication: |
435/006 ;
435/091.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Claims
What is claimed is:
1. A method for detecting a cancer in a brain tissue sample, the
method comprising the steps of: (A) providing the brain tissue
sample; and (B) analyzing the brain tissue sample for a Fra-1
marker.
2. The method of claim 1, wherein the step (B) of analyzing the
brain tissue sample comprises comparing the quantity of expression
of the Fra-1 marker to a first sample known to express detectable
levels of the Fra-1 marker and a second sample known to not express
detectable levels of the Fra-1 marker.
3. The method of claim 1, wherein the Fra-1 marker is a Fra-1
nucleic acid.
4. The method of claim 3, wherein the Fra-1 marker is an RNA.
5. The method of claim 3, wherein the Fra-1 nucleic acid is a
native Fra-1 nucleic acid.
6. The method of claim 3, wherein the step (A) of providing a
tissue sample comprises obtaining the brain tissue sample from a
human subject; and the step (B) of analyzing the brain tissue
sample comprises isolating RNA from the tissue sample, generating
cDNAs from the isolated RNA, amplifying the cDNAs by PCR to
generate a PCR product.
7. The method of claim 3, wherein the step (A) of providing a brain
tissue sample comprises obtaining the tissue sample from a human
subject; and the step (B) of analyzing the brain tissue sample
comprises isolating nucleic acid from the tissue sample, and
contacting the isolated nucleic acid with an oligonucleotide probe
that hybridizes under stringent hybridization conditions to the
Fra-1 nucleic acid.
8. The method of claim 7, wherein the oligonucleotide probe further
comprises a detectable label.
9. The method of claim 1, wherein the Fra-1 marker is a Fra-1
protein.
10. The method of claim 9, wherein the Fra-1 protein is a native
Fra-1 protein.
11. The method of claim 9, wherein the step (A) of providing a
brain tissue sample comprises obtaining the brain tissue sample
from a human subject; and the step (B) of analyzing the brain
tissue sample comprises contacting at least a portion of the brain
tissue sample with a probe that specifically binds to the Fra-1
protein.
12. The method of claim 11, wherein the probe comprises a
detectable label.
13. The method of claim 11, wherein the probe comprises an
antibody.
14. The method of claim 13, wherein the antibody is a polyclonal
antibody.
15. The method of claim 13, wherein the antibody is a monoclonal
antibody.
16. A method of modulating Fra-1 gene expression in a brain cancer
cell comprising the steps of: (A) providing a brain cancer cell
that expresses a Fra-1 gene; and (B) introducing into the cell an
agent that modulates the expression of the Fra-1 gene in the
cell.
17. The method of claim 16, wherein the agent is an
oligonucleotide.
18. The method of claim 16, wherein the agent is an antisense
oligonucleotide.
19. The method of claim 18, wherein the antisense oligonucleotide
hybridizes under stringent hybridization conditions to a
polynucleotide that encodes a Fra-1 protein.
20. A method of inhibiting VEGF-D gene expression in a brain cancer
cell comprising the steps of: (A) providing a brain cancer cell
that expresses a VEGF-D gene promoter and a Fra-1 protein; and (B)
introducing into the cell an agent that interferes with binding of
the Fra-1 protein to the VEGF-D gene promoter.
21. The method of claim 20, wherein the agent specifically binds a
c-Jun protein.
22. The method of claim 20, wherein the agent specifically binds
Fra-1 protein.
23. The method of claim 20, wherein the agent specifically binds
the VEGF-D promoter.
24. The method of claim 20, wherein the agent is a variant of a
native c-Jun protein that binds the Fra-1 protein but lacks the
ability to bind a VEGF-D gene promoter.
25. The method of claim 20, wherein the molecule is a variant of a
native Fra-1 protein that binds a c-Jun protein but lacks the
ability to bind a VEGF-D gene promoter.
26. The method of claim 20, wherein the step (B) of introducing an
agent that interferes with binding of the Fra-1 protein comprises
introducing an expression vector having a nucleic acid encoding the
agent into the cell.
27. The method of claim 26, wherein the agent is an antisense
oligonucleotide that hybridizes under stringent conditions to a
polynucleotide that encodes a Fra-1 protein.
28. The method of claim 26, wherein the agent is a variant of a
native c-Jun protein that binds the Fra-1 protein but lacks the
ability to bind a VEGF-D gene promoter.
29. The method of claim 26, wherein the agent is a variant of a
native Fra-1 protein that binds the c-Jun protein but lacks the
ability to bind a VEGF-D gene promoter.
30. The method of claim 20, wherein the brain cancer cell is
contained within the cranium of a human subject.
31. The method of claim 30, wherein the agent is administered to
the human subject by parenteral administration.
32. The method of claim 31, wherein the parenteral administration
is intravenous or intraarterial injection.
33. The method of claim 32, wherein the agent is introduced by
injection into the cranium of the human subject.
34. A method of identifying a test compound that modulates
expression of a Fra-1 gene in a brain cancer cell, the method
comprising the steps of: (A) providing a brain cancer cell
expressing a Fra-1 gene; (B) contacting the cell with the test
compound; and (C) detecting a modulation in the expression of the
Fra-1 gene, wherein detecting the modulation indicates that the
test compound modulates expression of the Fra-1 gene.
35. The method of claim 34, wherein the cell is derived from a
tissue sample isolated from a human brain.
36. The method of claim 34, wherein the step of detecting the
modulation in the expression of the Fra-1 gene comprises analyzing
the cell for a change in the amount of a Fra-1 marker in the
cell.
37. The method of claim 36, wherein the Fra-1 marker is a Fra-1
nucleic acid.
38. The method of claim 37, wherein the Fra-1 nucleic acid is an
RNA.
39. The method of claim 37, wherein the Fra-1 nucleic acid is a
native Fra-1 nucleic acid.
40. The method of claim 36, wherein the Fra-1 marker is a Fra-1
protein.
41. The method of claim 40, wherein the Fra-1 protein is a native
Fra-1 protein.
42. A method for inhibiting angiogenesis associated with a brain
cancer in a subject, the method comprising the steps of: (A)
providing an agent that interferes with Fra-1 binding to a VEGF-D
gene promoter; and (B) administering the agent to the central
nervous system of the subject in an amount effective to inhibit
blood vessel development associated with the brain cancer.
43. The method of claim 42, wherein the agent specifically binds a
c-Jun protein.
44. The method of claim 42, wherein the agent specifically binds a
Fra-1 protein
45. The method of claim 42, wherein the agent specifically binds
the VEGF-D gene promoter.
46. The method of claim 42, wherein the agent is a variant of a
native c-Jun protein that binds the Fra-1 protein but lacks the
ability to bind a VEGF-D gene promoter.
47. The method of claim 42, wherein the agent is a variant of a
native Fra-1 protein that binds a c-Jun protein but lacks the
ability to bind a VEGF-D gene promoter.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] The present application claims the priority of U.S.
provisional patent application No. 60/268,089 filed Feb. 12,
2001.
FIELD OF THE INVENTION
[0002] The invention relates to the fields of medicine,
angiogenesis and neuro-oncology. More particularly, the invention
relates to compositions and methods for detecting and treating
malignant tumors.
BACKGROUND OF THE INVENTION
[0003] Cancer is presently the second leading cause of death in
developed nations. Wingo et al., J. Reg. Management, 25:43-51
(1998). Despite recent research that has revealed many of the
molecular mechanisms of tumorigenesis, few new treatments have
achieved widespread clinical success in treating solid tumors.
Current treatments for most malignancies thus remain gross
resection, chemotherapy, and radiotherapy. While increasingly
successful, each of these treatments still causes numerous
undesired side effects. The primary cause of these side effects is
that none of these conventional methods specifically targets only
diseased cells. For example, surgery results in pain, traumatic
injury to healthy tissue, and scarring. Radiotherapy and
chemotherapy cause nausea, immune suppression, gastric ulceration
and secondary tumorigenesis.
[0004] In an effort to develop techniques to more specifically
target diseased cells, progress in tumor immunology has led to the
discovery of antigens that are preferentially or specifically
expressed by cancer cells. The identification of tumor-specific
cellular markers has proven extremely valuable for diagnosing and
assessing the progression of certain types of tumors. Antibodies
specific for tumor cell markers or ligands that bind specifically
to a tumor cell receptor have been successfully used in
diagnostics, including both the characterization of excised tissue
samples and in vivo imaging. Tumor-specific antibodies and ligands
have also been used in the targeted delivery of cytotoxic molecules
to specific tumor cells. Some tumor cell antigens are known to
function in the pathogenesis of a cancer. Modulating the function
of these antigens could impair the progression of the disease.
SUMMARY
[0005] The invention relates to the discovery that glioblastoma
multiforme (GBM) strongly expresses Fra-1, an AP-1 transcription
factor. The gene for VEGF-D, a vascular endothelial growth factor
that plays a role in angiogenesis, harbors an optimal AP-1 binding
site within its promoter region. When heterodimerized with c-Jun,
Fra-1 binds to the AP-1 site within the VEGF-D gene promoter and
activates expression of VEGF-D. Based on this discovery, central
nervous system (CNS) cancers such as GBM can be diagnosed and
treated using Fra-1 as a target tumor antigen. In addition, by
disrupting the interaction between Fra-1 and the VEGF-D gene
promoter, tumor angiogenesis can be inhibited.
[0006] In addition to playing a role in angiogenesis, Fra-1 and
other AP-1 regulated factors have been associated with tumor
invasiveness. AP-1 induced constitutive gene expression that occurs
in a transcription factor-specific manner in GBM contributes to the
high neo-vascularization and invasiveness of this fatal brain
tumor. Changes in a cell's phenotype due to expression of Fra-1,
including anchorage-independent growth and invasiveness, can be
evaluated in vitro. The motility of Fra-1 transfected cells may
also be analyzed in vitro. Furthermore, AP-1 activity in Fra-1
transfected cells can be measured using an artificial AP-1
dependent promoter. Thus, Fra-1 can also serve as a target for
inhibiting tumor invasiveness.
[0007] Accordingly, the invention features a method for detecting a
cancer in a brain tissue sample (e.g., one isolated from a human
subject). This method includes the steps of providing the brain
tissue sample; and analyzing the brain tissue sample for a Fra-1
marker such as a Fra-1 nucleic acid or Fra-1 protein. In this
method, the step of analyzing the brain tissue sample can include
comparing the quantity of expression of the Fra-1 marker to a first
sample known to express detectable levels of the Fra-1 marker (a
positive control) and a second sample known to not express
detectable levels of the Fra-1 marker (a negative control).
[0008] Fra-1 nucleic acid expression can be analyzed by isolating
RNA from the tissue sample, generating cDNAs from the isolated RNA,
amplifying the cDNAs by PCR to generate a PCR product.
Alternatively, Fra-1 nucleic acid expression can be analyzed by
isolating nucleic acid from the tissue sample, and contacting the
isolated nucleic acid with an oligonucleotide probe (e.g., a
labeled oligonucleotide probe) that hybridizes under stringent
hybridization conditions to the Fra-1 nucleic acid.
[0009] Fra-1 protein expression can be analyzed by contacting at
least a portion of the brain tissue sample with a probe that
specifically binds to the Fra-1 protein. The probe can be an
antibody (e.g., a polyclonal or monoclonal antibody), and can
include a detectable label.
[0010] In another aspect, the invention features a method of
modulating Fra-1 gene expression in a brain cancer cell. This
method includes the steps of: providing a brain cancer cell that
expresses a Fra-1 gene; and introducing into the cell an agent that
modulates the expression of the Fra-1 gene in the cell. The agent
can be an oligonucleotide such as an antisense oligonucleotide that
hybridizes under stringent hybridization conditions to a
polynucleotide that encodes a Fra-1 protein.
[0011] The invention also features a method of identifying a test
compound that modulates expression of a Fra-1 gene in a brain
cancer cell (e.g., one derived from a human brain). This method
includes the steps of: providing a brain cancer cell expressing a
Fra-1 gene; contacting the cell with the test compound; and
detecting a modulation in the expression of the Fra-1 gene.
Detecting the modulation indicates that the test compound modulates
expression of the Fra-1 gene. Modulation in the expression of the
Fra-1 gene can be assessed by analyzing the cell for a change in
the amount of a Fra-1 marker in the cell.
[0012] Also within the invention is a method for inhibiting
angiogenesis associated with a brain cancer in a subject. This
method includes the steps of: providing an agent that interferes
with Fra-1 binding to a VEGF-D gene promoter; and administering the
agent to the central nervous system of the subject in an amount
effective to inhibit blood vessel development associated with the
brain cancer. The agent that interferes with Fra-1 binding to a
VEGF-D gene promoter can be one that specifically binds a c-Jun
protein, a Fra-1 protein, or a Fra-1 gene promoter. The agent can
also be a variant of a native c-Jun protein that binds the Fra-1
protein but lacks the ability to bind a VEGF-D promoter; or a
variant of a native Fra-1 protein that binds a c-Jun protein but
lacks the ability to bind a VEGF-D promoter
[0013] Unless otherwise defined, all technical terms used herein
have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs. Commonly
understood definitions of molecular biology terms can be found in
Rieger et al., Glossary of Genetics: Classical and Molecular, 5th
edition, Springer-Verlag: New York, 1991; and Lewin, Genes V,
Oxford University Press: New York, 1994.
[0014] By the term "cancer" is meant any disorder of cell growth
that results in invasion and destruction of surrounding healthy
tissue by abnormal cells.
[0015] As used herein, the term "promoter" refers to the general
region of a DNA molecule that signals the start of transcription.
It is this region to which an RNA polymerase binds and initiates
transcription. Fra-1 protein, in concert with c-Jun protein, binds
the VEGF-D gene promoter. The VEGF-D gene promoter is described in
detail in Rocchigiani et al. Genomics 47:207-216 (1998). The VEGF-D
gene promoter of the sequence deposited with GenBank as Accession
No. Y12864 is denoted as nucleotides 1-465 (the exon 1
promoter).
[0016] As used herein, the term "vector" refers to a nucleic acid
molecule capable of transporting another nucleic acid to which it
has been linked. One type of preferred vector is an episome, i.e.,
a nucleic acid capable of extra-chromosomal replication. Preferred
vectors are those capable of autonomous replication and/or
expression of nucleic acids to which they are linked. Vectors
capable of directing the expression of genes to which they are
operatively linked are referred to herein as "expression
vectors."
[0017] A first nucleic acid sequence is "operably" linked with a
second nucleic acid sequence when the first nucleic acid sequence
is placed in a functional relationship with the second nucleic acid
sequence. For instance, a promoter is operably linked to a coding
sequence if the promoter affects the transcription or expression of
the coding sequence. Generally, operably linked nucleic acid
sequences are contiguous and, where necessary to join two protein
coding regions, in reading frame.
[0018] As used herein, the term "gene" means a nucleic acid
molecule that codes for a particular protein, or in certain cases,
a functional or structural RNA molecule. For example, a Fra-1 gene
encodes a Fra-1 protein. The phrase "nucleic acid" or a "nucleic
acid molecule" means a chain of two or more nucleotides such as RNA
(ribonucleic acid) and DNA (deoxyribonucleic acid). A "purified"
nucleic acid molecule is one that is substantially separated from
other nucleic acid sequences in a cell or organism in which the
nucleic acid naturally occurs (e.g., 30, 40, 50, 60, 70, 80, 90,
95, 96, 97, 98, 99, 100% free of contaminants). The term includes,
e.g., a recombinant nucleic acid molecule incorporated into a
vector, a plasmid, a virus, or a genome of a prokaryote or
eukaryote. Examples of purified nucleic acids include cDNAs,
fragments of genomic nucleic acids, nucleic acids produced
polymerase chain reaction (PCR), nucleic acids formed by
restriction enzyme treatment of genomic nucleic acids, recombinant
nucleic acids, and chemically synthesized nucleic acid molecules. A
"recombinant" nucleic acid molecule is one made by an artificial
combination of two otherwise separated segments of sequence, e.g.,
by chemical synthesis or by the manipulation of isolated segments
of nucleic acids by genetic engineering techniques.
[0019] The phrases "Fra-1 gene," "Fra-1 polynucleotide," or "Fra-1
nucleic acid" as used herein mean a native Fra-1-encoding nucleic
acid sequence, e.g., the native human (Genbank Accession Nos.
X16707 and D14493), rat (Accession Nos. V24154 and M19651), and
mouse (Accession Nos. U34245 and AF017128) Fra-1 genes; a native
form Fra-1 cDNA; a nucleic acid having sequences from which a Fra-1
cDNA can be transcribed; and/or allelic variants and homologs of
the foregoing. The terms encompass double-stranded DNA,
single-stranded DNA, and RNA.
[0020] As used herein, "protein" or "polypeptide" mean any
peptide-linked chain of amino acids, regardless of length or
post-translational modification, e.g., glycosylation or
phosphorylation. A "purified" polypeptide is one that is
substantially separated from other polypeptides in a cell or
organism in which the polypeptide naturally occurs (e.g., 30, 40,
50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% free of
contaminants).
[0021] By the phrase "Fra-1 protein" or "Fra-1 polypeptide" is
meant an expression product of a Fra-1 gene such as a native Fra-1
protein, or a protein that shares at least 65% (but preferably 75,
80, 85, 90 , 95, 96, 97 , 98, or 99%) amino acid sequence identity
with one of the foregoing and displays a functional activity of a
human native Fra-1 protein. A "functional activity" of a protein is
any activity associated with the physiological function of the
protein. For example, functional activities of a native Fra-1
protein may include binding c-Jun, binding a VEGF-D gene promoter,
activating the VEGF-D gene, increased expression in certain
neoplastic tissues, and the ability to stimulate angiogenesis.
[0022] When referring to a nucleic acid molecule or polypeptide,
the term "native" refers to a naturally-occurring (e.g., a
"wild-type") nucleic acid or polypeptide. A "homolog" of a Fra-1
gene from one species of organism is a gene sequence encoding a
Fra-1 polypeptide isolated from an organism of a different species.
Similarly, a "homolog" of a native Fra-1 polypeptide is an
expression product of a Fra-1 gene homolog.
[0023] As used herein, a "Fra-1 marker" is any molecule whose
presence in a sample (e.g., a cell) indicates that a Fra-1 gene is
expressed in the sample. Fra-1 markers include Fra-1 nucleic acids
and Fra-1 proteins. "Expressing a Fra-1 gene" or like phrases mean
that a sample contains a transcription product (e.g., messenger
RNA, i.e., "mRNA") of a Fra-1 gene or a translation product of a
Fra-1 protein-encoding nucleic acid (e.g., a Fra-1 protein). A cell
expresses a Fra-1 gene when it contains a detectable level of a
Fra-1 nucleic acid or a Fra-1 protein.
[0024] A "fragment" of a Fra-1 nucleic acid is a portion of a Fra-1
nucleic acid that is less than full-length and comprises at least a
minimum length capable of hybridizing specifically with a native
Fra-1 nucleic acid under stringent hybridization conditions. The
length of such a fragment is preferably at least 15 nucleotides,
more preferably at least 20 nucleotides, and most preferably at
least 30 nucleotides of a native Fra-1 nucleic acid sequence. A
"fragment" of a Fra-1 polypeptide is a portion of a Fra-1
polypeptide that is less than full-length (e.g., a polypeptide
consisting of 5, 10, 15, 20, 30, 40, 50, 75, 100 or more amino
acids of a native Fra-1 protein), and preferably retains at least
one functional activity of a native Fra-1 protein.
[0025] When referring to hybridization of one nucleic acid to
another, "low stringency conditions" means in 10% formamide,
5.times. Denhart's solution, 6.times.SSPE, 0.2% SDS at 42.degree.
C., followed by washing in 1.times.SSPE, 0.2% SDS, at 50.degree.
C.; "moderate stringency conditions" means in 50% formamide,
5.times. Denhart's solution, 5.times.SSPE, 0.2% SDS at 42.degree.
C., followed by washing in 0.2.times.SSPE, 0.2% SDS, at 65.degree.
C.; and "high stringency conditions" means in 50% formamide,
5.times. Denhart's solution, 5.times.SSPE, 0.2% SDS at 42.degree.
C., followed by washing in 0.1.times.SSPE, and 0.1% SDS at
65.degree. C. The phrase "stringent hybridization conditions" means
low, moderate, or high stringency conditions.
[0026] By the term "Fra-1-specific antibody" is meant an antibody
that binds a Fra-1 protein and displays no substantial binding to
other naturally occurring proteins other than those sharing the
same antigenic determinants as the Fra-1 protein. The term includes
polyclonal and monoclonal antibodies as well as antibody fragments.
As used herein, "bind," "binds," or "interacts with" means that one
molecule recognizes and adheres to a particular second molecule in
a sample, but does not substantially recognize or adhere to other
structurally unrelated molecules in the sample. Generally, a first
molecule that "specifically binds" a second molecule has a binding
affinity greater than about 10.sup.5 to 10.sup.6 moles/liter for
that second molecule.
[0027] The term "labeled," with regard to a probe or antibody, is
intended to encompass direct labeling of the probe or antibody by
coupling (i.e., physically linking) a detectable substance to the
probe or antibody.
[0028] Although methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
the present invention, suitable methods and materials are described
below. All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety. In the case of conflict, the present specification,
including definitions will control. The particular embodiments
discussed below are illustrative only and not intended to be
limiting.
DETAILED DESCRIPTION
[0029] The invention provides methods and compositions for
diagnosing and treating malignant tumors including, in particular,
brain cancers such as GBM. For example, according to the invention,
brain cancer is diagnosed by analyzing a brain tissue sample for
expression of a Fra-1 marker, such as a Fra-1 nucleic acid or a
Fra-1 protein. Brain cancer is treated by introducing into the
cancer cells an agent that modulates Fra-1 gene expression in the
cells. A brain cancer can also be treated by inhibiting the
angiogenesis associated with the cancer by interfering with Fra-1
activation of a VEGF-D gene, e.g., by administering a molecule that
interferes with the Fra-1/VEGF-D promoter or the Fra-1/c-Jun
interaction to a subject suffering from a brain cancer. The
invention also provides a method for identifying a test compound
that modulates expression of a Fra-1 gene in a brain cancer cell.
To identify such a compound, a brain cancer cell expressing a Fra-1
gene is contacted with a test compound and analyzed for modulations
in Fra-1 expression.
[0030] The below described preferred embodiments illustrate
adaptations of these compositions and methods. Nonetheless, from
the description of these embodiments, other aspects of the
invention can be made and/or practiced based on the description
provided below.
Biological Methods
[0031] Methods involving conventional molecular biology techniques
are described herein. Such techniques are generally known in the
art and are described in detail in methodology treatises such as
Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed.
Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989; and Current Protocols in Molecular Biology, ed.
Ausubel et al., Greene Publishing and Wiley-Interscience, New York,
1992 (with periodic updates). Various techniques using polymerase
chain reaction (PCR) are described, e.g., in Innis et al., PCR
Protocols: A Guide to Methods and Applications, Academic Press: San
Diego, 1990. PCR-primer pairs can be derived from known sequences
by known techniques such as using computer programs intended for
that purpose (e.g., Primer, Version 0.5, .COPYRGT.1991, Whitehead
Institute for Biomedical Research, Cambridge, Mass.). Methods for
chemical synthesis of nucleic acids are discussed, for example, in
Beaucage and Carruthers, Tetra. Letts. 22:1859-1862, 1981, and
Matteucci et al., J. Am. Chem. Soc. 103:3185, 1981. Chemical
synthesis of nucleic acids can be performed, for example, on
commercial automated oligonucleotide synthesizers. Immunological
methods (e.g., preparation of antigen-specific antibodies,
immunoprecipitation, and immunoblotting) are described, e.g., in
Current Protocols in Immunology, ed. Coligan et al., John Wiley
& Sons, New York, 1991; and Methods of Immunological Analysis,
ed. Masseyeffet al., John Wiley & Sons, New York, 1992.
Conventional methods of gene transfer and gene therapy can also be
adapted for use in the present invention. See, e.g., Gene Therapy:
Principles and Applications, ed. T. Blackenstein, Springer Verlag,
1999; Gene Therapy Protocols (Methods in Molecular Medicine), ed.
P. D. Robbins, Humana Press, 1997; and Retro-vectors for Human Gene
Therapy, ed. C. P. Hodgson, Springer Verlag, 1996.
Method of Detecting a Cancer
[0032] The invention provides a method for detecting a cancer in a
brain tissue sample by analyzing the brain tissue sample for a
Fra-1 marker such as a Fra-1 nucleic acid or Fra-1 protein. A
preferred version of this method includes comparing the quantity of
Fra-1 marker expression in the brain tissue sample to one or more
control samples. The control samples can be a positive control
sample, i.e., a sample known to express detectable levels of the
Fra-1 marker using the same method of analysis as used for the
brain tissue sample; and a negative control sample, i.e., a sample
known not to express detectable levels of the Fra-1 marker using
the same method of analysis as used for the brain tissue sample.
Use of positive and negative controls ensures accuracy of test
results.
Cancerous Tumors
[0033] The invention is based on the discovery that brain cancer
cells express higher levels of Fra-1 than do normal brain cells and
that Fra-1 activates VEGF-D gene expression. Accordingly, preferred
methods of the invention involve analyzing the brain cancer cells,
particularly glioma and GBM cells, for Fra-1 expression. Various
forms of glioma/GBM are described in more detail in Dai and
Holland, Biochim. Biophys. Acta, 1551:M19-27, 2001 and Holland,
Nat. Rev. Genetics, 2:120-129, 2001. In addition to brain cancers,
the methods and compositions described herein might be used with
other types of cancers that express high levels of Fra-1 and
VEGF-D.
Brain Tissue Samples
[0034] The invention provides methods for analyzing a brain tissue
sample and administering a composition to a brain cancer in a
mammal. Surgical techniques for obtaining brain tissue samples as
well as administering various compositions to the brain are well
known in the art. For example, such methods are described in
standard neuro-surgery texts such as Atlas of Neurosurgery: Basic
Approaches to Cranial and Vascular Procedures, by F. Meyer,
Churchill Livingstone, 1999; Stereotactic and Image Directed
Surgery of Brain Tumors, 1st ed., by David G. T. Thomas, W B
Saunders Co., 1993; and Cranial Microsurgery: Approaches and
Techniques, by L. N. Sekhar and E. De Oliveira, 1st ed., Thieme
Medical Publishing, 1999. Methods for obtaining and analyzing brain
tissue are also described in Belay et al., Arch. Neurol. 58:
1673-1678 (2001); and Seijo et al., J. Clin. Microbiol. 38:
3892-3895 (2000).
Detection of Fra-1 Polynucleotides and Proteins
[0035] The invention encompasses methods for detecting the presence
of a Fra-1 marker such as a Fra-1 protein or a Fra-1 nucleic acid
in a biological sample as well as methods for measuring the level
of a Fra-1 marker in a biological sample. Such methods are useful
for diagnosing cancer associated with Fra-1 expression, e.g., brain
cancer.
[0036] An exemplary method for detecting the presence or absence of
a Fra-1 protein or nucleic acid in a biological sample involves
obtaining a biological sample from a subject (e.g., a human
patient), contacting the biological sample with a compound or an
agent capable of detecting a Fra-1 protein or a nucleic acid
encoding a Fra-1 protein (e.g., antibody, mRNA or genomic DNA), and
analyzing binding of the compound or agent to the sample after
washing. Those samples having specifically bound compound or agent
express a Fra-1 protein or a nucleic acid encoding a Fra-1
protein.
[0037] A preferred agent for detecting a nucleic acid encoding a
Fra-1 protein is a labeled nucleic acid probe capable of
hybridizing to the nucleic acid encoding the Fra-1 protein. The
nucleic acid probe can be, for example, all or a portion of a Fra-1
gene itself or all or a portion of a complement of a Fra-1 gene.
Similarly, the probe can also be all or a portion of a Fra-1 gene
variant, or all or a portion of a complement of a Fra-1 gene
variant. For instance, oligonucleotides at least 15, 30, 50, 100,
250, or 500 nucleotides in length that specifically hybridize under
stringent conditions to a native Fra-1 nucleic acid or a complement
of a native Fra-1 nucleic acid can be used as probes within the
invention. A preferred agent for detecting a Fra-1 protein is an
antibody capable of binding to a Fra-1 protein, preferably an
antibody with a detectable label. Such antibodies can be
polyclonal, or more preferably, monoclonal. An intact antibody, or
a fragment thereof (e.g., Fab or F(ab').sub.2) can be used.
[0038] Methods of the invention can be used to detect an mRNA
encoding a Fra-1 protein, a genomic DNA encoding a Fra-1 protein,
or a Fra-1 protein in a biological sample in vitro as well as in
vivo. The quantity of expression of Fra-1 marker from a brain
tissue sample may be compared with appropriate controls such as a
first sample known to express detectable levels of the Fra-1 marker
(i.e., a positive control) and a second sample known to not express
detectable levels of the Fra-1 marker (i.e., a negative control).
For example, in vitro techniques for detection of mRNAs encoding a
Fra-1 protein include PCR amplification methods, Northern
hybridizations, and in situ hybridizations. In vitro techniques for
detection of a Fra-1 protein include enzyme linked immunosorbent
assays (ELISAs), Western blots, immunoprecipitations and
immunofluorescence. In vitro techniques for detection of genomic
DNA encoding Fra-1 include Southern hybridizations. Furthermore, in
vivo techniques for detection of a Fra-1 protein include
introducing a labeled anti-Fra-1 antibody into a biological sample
or test subject. For example, the antibody can be labeled with a
radioactive marker whose presence and location in a biological
sample or test subject can be detected by standard imaging
techniques.
[0039] Myriad detectable labels that may be used in a diagnostic
assay for Fra-1 expression are known in the art. Nucleic acid
probes, for example, may be labeled with chemiluminescent or
radioactive substance. The amount of labeled probe bound to a Fra-1
marker may then be assessed using photographic or X-ray film or
other suitable methods for detecting luminescence or radioactivity.
Antibodies used in methods for detecting Fra-1 protein may be
conjugated to a detectable label, e.g., an enzyme such as
horseradish peroxidase. Antibodies labeled with horseradish
peroxidase can be detected by adding an appropriate substrate that
produces a color change in the presence of horseradish peroxidase.
Several other detectable labels that may be used are known. Common
examples of these include alkaline phosphatase, horseradish
peroxidase, fluorescent compounds, luminescent compounds, colloidal
gold, magnetic particles, biotin, radioisotopes, and enzymes.
Nucleic Acids Encoding Fra-1 Proteins
[0040] Methods of the present invention relate to Fra-1 nucleic
acids. Preferred nucleic acid molecules for use in the invention
include native human (Genbank Accession Nos. X16707 and D14493),
rat (U24154 and M19651), and mouse (U34245 and AF017128) Fra-1
polynucleotides. Nucleic acid molecules utilized in the present
invention may be in the form of RNA or in the form of DNA (e.g.,
cDNA, genomic DNA, and synthetic DNA). The DNA may be
double-stranded or single-stranded, and if single-stranded may be
the coding (sense) strand or non-coding (anti-sense) strand. In
addition to a coding sequence which encodes a native Fra-1 protein,
other nucleic acid molecules that can be used in the invention
include variants of a native Fra-1 gene such as those that encode
fragments, analogs and derivatives of a native Fra-1 protein. Such
variants may be, e.g., a naturally occurring allelic variant of a
native Fra-1 gene or a homolog of a native Fra-1 gene.
[0041] Vectors encoding a native or variant Fra-1, Fra-1 binding
species, c-Jun binding species, or antisense construct can be
generated by recombinant DNA technology methods that are known in
the art. Suitable vectors include plasmid vectors, viral vectors,
or other types of vectors known or newly discovered in the art. The
criterion for use is only that the vector be capable of replicating
and expressing a native or variant Fra-1 protein, Fra-1 binding
species, c-Jun binding species, or antisense construct sequence.
Expression of the sequence encoding native a or variant Fra-1
protein, Fra-1 binding species, c-Jun binding species, or antisense
construct can be directed by any promoter known in the art to act
in mammalian, and preferably in human, cells. Such promoters can be
inducible or constitutively active and include but are not limited
to: the SV40 early promoter region (Bernoist et al., Nature
290:304, 1981); the promoter contained in the 3' long terminal
repeat of Rous Sarcoma virus (Yamamoto et al., Cell 22:787-797,
1988); the herpes thymidine kinase promoter (Wagner et al., PNAS
78:1441, 1981); or the regulatory sequences of the metallothionein
gene (Brinster et al., Nature 296:39, 1988).
[0042] Vectors utilized in methods of the invention to deliver a
native or variant Fra-1 protein, Fra-1 binding species, c-Jun
binding species, or antisense construct may also contain, if
desired, regulatory elements such as a tissue-specific promoter or
enhancer, a transcription initiation start site, a ribosomal
binding site, an RNA processing signal, a transcription termination
site, and/or a polyadenylation signal. Tissue-specific promoters
and enhancers used in methods of the invention may include those
that direct gene expression specifically in CNS tissue, and more
preferably in cells of astrocyte lineage. An example of a neuron
and astrocyte-specific promoter is the proximal region of the c-fos
promoter. Onteniente et al., Brain Res. Mol. Brain Res. 21:225-234
(1994). Another example of a promoter that directs
astrocyte-specific gene expression is the human glial fibrillary
acidic protein (hGFAP) promoter. Ding et al., Cancer Res. 61:
3826-3836 (2001); and Vandier et al., Cancer Gene Therapy
7:1120-1126 (2000). Examples of enhancers that direct high-level
and specific gene expression in astrocytes are two apoE gene
enhancers that are located downstream of the apoE gene. Greham et
al., J. Neurosci. 21: 812-822 (2001).
Probes and Primers
[0043] Nucleic acids that hybridize under stringent conditions to
Fra-1 nucleic acid or the complement of Fra-1 nucleic acid can be
used in the invention. For example, such nucleic acids can be those
that hybridize to Fra-1 nucleic acid or the complement of a Fra-1
nucleic acid under low stringency conditions, moderate stringency
conditions, or high stringency conditions. Preferred such nucleic
acids are those having a nucleotide sequence that is the complement
of all or a portion of Fra-1 nucleic acid. Others that might be
used include polynucleotides that share at least 65% (e.g., 65, 70,
75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, and 99%) sequence
identity to a native Fra-1 nucleic acid or the complement of Fra-1
nucleic acid. Nucleic acids that hybridize under stringent
conditions to or share at least 65% sequence identity with Fra-1
nucleic acid or the complement of Fra-1 nucleic acid can be
obtained by techniques known in the art such as by making mutations
in a native Fra-1 gene, or by isolation from an organism expressing
such a nucleic acid (e.g., an allelic variant).
[0044] Methods of the invention utilize oligonucleotide probes
(i.e., isolated nucleic acid molecules conjugated with a detectable
label or reporter molecule, e.g., a radioactive isotope, ligand,
chemiluminescent agent, or enzyme); and oligonucleotide primers
(i.e., isolated nucleic acid molecules that can be annealed to a
complementary target DNA strand by nucleic acid hybridization to
form a hybrid between the primer and the target DNA strand, then
extended along the target DNA strand by a polymerase, e.g., a DNA
polymerase). Primer pairs can be used for amplification of a
nucleic acid sequence, e.g., by the polymerase chain reaction (PCR)
or other conventional nucleic-acid amplification methods.
[0045] PCR primers can be used to amplify Fra-1 nucleic acids using
known PCR and RT-PCR protocols. Such primers can be designed
according to known methods as PCR primer design is generally known
in the art. See, e.g., methodology treatises such as Basic Methods
in Molecular Biology, 2nd ed., ed. Davis et al., Appleton &
Lange, Norwalk, Conn., 1994; and Molecular Cloning: A Laboratory
Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989. PCR primer pairs
previously used to amplify a Fra-1 nucleic acid sequence are
described in Hu et al., Clin. Cancer Res. 7:2213-2221 (2001); and
Chiappetta et al., Clin. Cancer Res. 6:4300-4306 (2000).
[0046] Probes and primers utilized in methods of the invention are
generally 15 nucleotides or more in length, preferably 20
nucleotides or more, more preferably 25 nucleotides, and most
preferably 30 nucleotides or more. Preferred probes and primers are
those that hybridize to a native Fra-1 gene (or cDNA or mRNA)
sequence under high stringency conditions, and those that hybridize
to Fra-1 gene homologs under at least moderately stringent
conditions. Preferably, probes and primers according to the present
invention have complete sequence identity with a native Fra-1
nucleic acid sequence. However, probes differing from this sequence
that retain the ability to hybridize to a native Fra-1 gene
sequence under stringent conditions may be designed by conventional
methods and used in the invention. Primers and probes based on the
Fra-1 gene sequences disclosed herein can be used to confirm (and,
if necessary, to correct) the disclosed Fra-1 gene sequences by
conventional methods, e.g., by re-cloning and sequencing a native
Fra-1 gene or cDNA.
Fra-1 Proteins
[0047] The invention also provides methods involving Fra-1
proteins. Fra-1, also known as fos-related antigen-1, is found to
be expressed at higher levels in GBM cells, such as G48a, when
compared with normal human tissue. The Fra-1 protein is
approximately 42 kDa and is localized to the nucleus, which would
be expected for a transcription factor.
[0048] Methods of the present invention may utilize a purified
Fra-1 protein encoded by a nucleic acid of the invention. A
preferred form of Fra-1 is a purified native human Fra-1 protein
that has the amino acid sequence deposited with NCBI as accession
No. NP005429. Other forms of Fra-1 include those of mouse
(SwissProt accession No. P48755) and rat (SwissProt accession No.
P10158).
[0049] Variants of native Fra-1 proteins such as fragments, analogs
and derivatives of native Fra-1 proteins may also be used in
methods of the invention. Such variants include, e.g., a
polypeptide encoded by a naturally occurring allelic variant of a
native Fra-1 gene, a polypeptide encoded by an alternative splice
form of a native Fra-1 gene, a polypeptide encoded by a homolog of
a native Fra-1 gene, and a polypeptide encoded by a non-naturally
occurring variant of a native Fra-1 gene.
[0050] Fra-1 protein variants have a peptide sequence that differs
from a native Fra-1 protein in one or more amino acids. The peptide
sequence of such variants can feature a deletion, addition, or
substitution of one or more amino acids of a native Fra-1
polypeptide. Amino acid insertions are preferably of about 1 to 4
contiguous amino acids, and deletions are preferably of about 1 to
10 contiguous amino acids. In some applications, variant Fra-1
proteins substantially maintain a native Fra-1 protein functional
activity (e.g., association with cancer or ability to modulate
angiogenesis). For other applications, variant Fra-1 proteins lack
or feature a significant reduction in a Fra-1 protein functional
activity. Where it is desired to retain a functional activity of
native Fra-1 protein, preferred Fra-1 protein variants can be made
by expressing nucleic acid molecules within the invention that
feature silent or conservative changes. Variant Fra-1 proteins with
substantial changes in functional activity can be made by
expressing nucleic acid molecules within the invention that feature
less than conservative changes.
[0051] Fra-1 protein fragments corresponding to one or more
particular motifs and/or domains or to arbitrary sizes, for
example, at least 5, 10, 25, 50, 75, 100, 125, 150, 175, 200, and
250 amino acids in length may be utilized in methods of the present
invention. Isolated peptidyl portions of Fra-1 proteins can be
obtained by screening peptides recombinantly produced from the
corresponding fragment of the nucleic acid encoding such peptides.
In addition, fragments can be chemically synthesized using
techniques known in the art such as conventional Merrifield solid
phase f-Moc or t-Boc chemistry. For example, a Fra-1 protein used
in methods of the present invention may be arbitrarily divided into
fragments of desired length with no overlap of the fragments, or
preferably divided into overlapping fragments of a desired length.
The fragments can be produced (recombinantly or by chemical
synthesis) and tested to identify those peptidyl fragments which
can function as either agonists or antagonists of a native Fra-1
protein.
[0052] Methods of the invention may also involve recombinant forms
of the Fra-1 proteins. Recombinant polypeptides preferred by the
present invention, in addition to native Fra-1 protein, are encoded
by a nucleic acid that has at least 85% sequence identity (e.g.,
85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%)
with a native Fra-1 nucleic acid sequence. In a preferred
embodiment, variant Fra-1 proteins lack one or more functional
activities of native Fra-1 protein (e.g., binding c-Jun and
activating VEGF-D gene expression).
[0053] Fra-1 protein variants can be generated through various
techniques known in the art. For example, Fra-1 protein variants
can be made by mutagenesis, such as by introducing discrete point
mutation(s), or by truncation. Mutation can give rise to a Fra-1
protein variant having substantially the same, or merely a subset
of the functional activity of a native Fra-1 protein.
Alternatively, antagonistic forms of the protein can be generated
which are able to inhibit the function of the naturally occurring
form of the protein, such as by competitively binding to another
molecule that interacts with Fra-1 protein. In addition, agonistic
forms of the protein may be generated that constitutively express
one or more Fra-1 functional activities. Other variants of Fra-1
proteins that can be generated include those that are resistant to
proteolytic cleavage, as for example, due to mutations that alter
protease target sequences. Whether a change in the amino acid
sequence of a peptide results in a Fra-1 protein variant having one
or more functional activities of a native Fra-1 protein can be
readily determined by testing the variant for a native Fra-1
protein functional activity.
[0054] Nucleic acid molecules encoding Fra-1 fusion proteins may be
used in methods of the invention. Such nucleic acids can be made by
preparing a construct (e.g., an expression vector) that expresses a
Fra-1 fusion protein when introduced into a suitable host. For
example, such a construct can be made by ligating a first
polynucleotide encoding a Fra-1 protein fused in frame with a
second polynucleotide encoding another protein such that expression
of the construct in a suitable expression system yields a fusion
protein.
[0055] As another example, Fra-1 protein variants can be generated
from a degenerate oligonucleotide sequence. Chemical synthesis of a
degenerate gene sequence can be carried out in an automatic DNA
synthesizer, and the synthetic genes then ligated into an
appropriate expression vector. The purpose of a degenerate set of
genes is to provide, in one mixture, all of the sequences encoding
the desired set of potential Fra-1 protein sequences. The synthesis
of degenerate oligonucleotides is well known in the art (see for
example, Narang, S A (1983) Tetrahedron 39:3; Itakura et al. (1981)
Recombinant DNA, Proc 3rd Cleveland Sympos. Macromolecules, ed. A G
Walton, Amsterdam: Elsevier pp 273-289; Itakura et al. (1984) Annu.
Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike
et al. (1983) Nucleic Acid Res. 11:477. Such techniques have been
employed in the directed evolution of other proteins (see, for
example, Scott et al. (1990) Science 249:386-390; Roberts et al.
(1992) Proc. Natl. Acad. Sci. USA 89:2429-2433; Devlin et al.
(1990) Science 249: 404-406; Cwirla et al. (1990) Proc. Natl. Acad.
Sci. USA 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409;
5,198,346; and 5,096,815).
[0056] Similarly, a library of coding sequence fragments can be
provided for a Fra-1 gene clone in order to generate a variegated
population of Fra-1 protein fragments for screening and subsequent
selection of fragments having one or more native Fra-1 protein
functional activities. A variety of techniques are known in the art
for generating such libraries, including chemical synthesis. In one
embodiment, a library of coding sequence fragments can be generated
by (i) treating a double-stranded PCR fragment of a Fra-1 gene
coding sequence with a nuclease under conditions wherein nicking
occurs only about once per molecule; (ii) denaturing the
double-stranded DNA; (iii) renaturing the DNA to form
double-stranded DNA which can include sense/antisense pairs from
different nicked products; (iv) removing single-stranded portions
from reformed duplexes by treatment with S1 nuclease; and (v)
ligating the resulting fragment library into an expression vector.
By this exemplary method, an expression library can be derived
which codes for N-terminal, C-terminal and internal fragments of
various sizes.
[0057] A wide range of techniques are known in the art for
screening gene products of combinatorial libraries made by point
mutations or truncation, and for screening cDNA libraries for gene
products having a certain property. Such techniques will be
generally adaptable for rapid screening of the gene libraries
generated by the combinatorial mutagenesis of Fra-1 gene variants.
The most widely used techniques for screening large gene libraries
typically involve cloning the gene library into replicable
expression vectors, transforming appropriate cells with the
resulting library of vectors, and expressing the combinatorial
genes under conditions in which detection of a desired activity
facilitates relatively easy isolation of the vector encoding the
gene whose product was detected.
[0058] Combinatorial mutagenesis has a potential to generate very
large libraries of mutant proteins, e.g., in the order of 10.sup.26
molecules. To screen a large number of protein mutants, techniques
that allow one to avoid the very high proportion of non-functional
proteins in a random library and simply enhance the frequency of
functional proteins (thus decreasing the complexity required to
achieve a useful sampling of sequence space) can be used. For
example, recursive ensemble mutagenesis (REM), an algorithm that
enhances the frequency of functional mutants in a library when an
appropriate selection or screening method is employed, might be
used. Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA
89:7811-7815; Yourvan et al. (1992) Parallel Problem Solving from
Nature, 2., In Maenner and Manderick, eds., Elsevier Publishing
Co., Amsterdam, pp. 401-410; Delgrave et al. (1993) Protein
Engineering 6(3):327-331.
[0059] Methods of the invention may utilize mimetics, e.g. peptide
or non-peptide agents, that are able to disrupt binding of a Fra-1
protein to other proteins or molecules with which a native Fra-1
protein interacts. Thus, the mutagenic techniques described herein
can also be used to map which determinants of Fra-1 protein
participate in the intermolecular interactions involved in, for
example, binding of a Fra-1 protein to other proteins which may
function upstream (e.g., activators or repressors of Fra-1
functional activity) of the Fra-1 protein or to proteins or nucleic
acids which may function downstream of the Fra-1 protein (e.g.
VEGF-D promoter), and whether such molecules are positively or
negatively regulated by the Fra-1 protein. To illustrate, the
critical residues of a Fra-1 protein which are involved in
molecular recognition of, for example, the Fra-1 protein or other
components upstream or downstream of the Fra-1 protein can be
determined and used to generate Fra-1 protein-derived
peptidomimetics which competitively inhibit binding of the Fra-1
protein to that moiety. By employing scanning mutagenesis to map
the amino acid residues of a Fra-1 protein that are involved in
binding other proteins (e.g., c-Jun), peptidomimetic compounds can
be generated which mimic those residues of a native Fra-1 protein.
Such mimetics may then be used to interfere with the normal
function of a Fra-1 protein. For instance, non-hydrolyzable peptide
analogs of such residues can be generated using benzodiazepine
(e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G.
R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988),
azepine (e.g., see Huffman et al. in Peptides: Chemistry and
Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands,
1988), substituted gamma lactam rings (Garvey et al. in Peptides:
Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,
Netherlands, 1988), keto-methylene pseudopepitides (Ewenson et al.
(1986) J. Med. Chem. 29:295; and Ewenson et al. in Peptides:
Structure and Function (Proceedings of the 9th American Peptide
Symposium) Pierce Chemical Co. Rockland, Ill, 1985), eta-turn
dipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and
Sato et al. (1986) J. Chem. Soc. Perkin. Trans. 1:1231), and
beta-aminoalcohols (Gordon et al. (1985) Biochem. Biophys. Res.
Commun. 126:419; and Dann et al. (1986) Biochem. Biophys. Res.
Commun. 134:71). Fra-1 proteins may also be chemically modified to
create Fra-1 protein derivatives by forming covalent or aggregate
conjugates with other chemical moieties, such as glycosyl groups,
lipids, phosphate, acetyl groups and the like. Covalent derivatives
of Fra-1 protein can be prepared by linking the chemical moieties
to functional groups on amino acid side chains of the protein or at
the N-terminus or at the C-terminus of the polypeptide.
Antibodies
[0060] Antibodies that specifically bind Fra-1 proteins can be used
in methods of the invention, for example, in the detection of Fra-1
protein markers. Polyclonal antibodies are heterogeneous
populations of antibody molecules that are contained in the sera of
immunized animals. Antibodies used in methods of the invention
include polyclonal antibodies and, in addition, monoclonal
antibodies, single chain antibodies, Fab fragments, F(ab').sub.2
fragments, and molecules produced using a Fab expression library.
Monoclonal antibodies, which are homogeneous populations of
antibodies to a particular antigen, can be prepared using the Fra-1
proteins described above and standard hybridoma technology (see,
for example, Kohler et al., Nature 256:495, 1975; Kohler et al.,
Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol.
6:292, 1976; Hammerling et al., "Monoclonal Antibodies and T Cell
Hybridomas," Elsevier, N.Y., 1981; Ausubel et al., supra). In
particular, monoclonal antibodies can be obtained by any technique
that provides for the production of antibody molecules by
continuous cell lines in culture such as described in Kohler et
al., Nature 256:495, 1975, and U.S. Pat. No. 4,376,110; the human
B-cell hybridoma technique (Kosbor et al., Immunology Today 4:72,
1983; Cole et al., Proc. Natl. Acad. Sci. USA 80:2026, 1983), and
the EBV-hybridoma technique (Cole et al., "Monoclonal Antibodies
and Cancer Therapy," Alan R. Liss, Inc., pp. 77-96, 1983). Such
antibodies can be of any immunoglobulin class including IgG, IgM,
IgE, IgA, IgD and any subclass thereof.
[0061] Antibodies that specifically recognize and bind to Fra-1 are
useful in methods of the present invention. For example, such
antibodies can be used in an immunoassay to monitor the level of a
Fra-1 protein produced by a mammal (e.g., to determine the amount
or subcellular location of a Fra-1 protein). Methods of the
invention may also utilize antibodies, for example, in the
detection of a Fra-1 protein in a biological sample. Antibodies
also can be used in a screening assay to measure the effect of a
candidate compound on expression or localization of a Fra-1
protein.
Modulating Fra-1 Expression
Antisense, Ribozyme, Triplex Techniques
[0062] Another aspect of the invention relates to the use of
purified antisense nucleic acids to inhibit expression of Fra-1.
Antisense nucleic acid molecules within the invention are those
that specifically hybridize (e.g. bind) under cellular conditions
to cellular mRNA and/or genomic DNA encoding a Fra-1 protein in a
manner that inhibits expression of the Fra-1 protein, e.g., by
inhibiting transcription and/or translation. The binding may be by
conventional base pair complementarity, or, for example, in the
case of binding to DNA duplexes, through specific interactions in
the major groove of the double helix.
[0063] Antisense constructs can be delivered, for example, as an
expression plasmid which, when transcribed in the cell, produces
RNA which is complementary to at least a unique portion of the
cellular mRNA which encodes a Fra-1 protein. Alternatively, the
antisense construct can take the form of an oligonucleotide probe
generated ex vivo which, when introduced into a Fra-1 protein
expressing cell, causes inhibition of Fra-1 protein expression by
hybridizing with an mRNA and/or genomic sequences coding for Fra-1
protein. Such oligonucleotide probes are preferably modified
oligonucleotides that are resistant to endogenous nucleases, e.g.
exonucleases and/or endonucleases, and are therefore stable in
vivo. Exemplary nucleic acid molecules for use as antisense
oligonucleotides are phosphoramidate, phosphothioate and
methylphosphonate analogs of DNA (see, e.g., U.S. Pat. Nos.
5,176,996; 5,264,564; and 5,256,775). Additionally, general
approaches to constructing oligomers useful in antisense therapy
have been reviewed, for example, by Van der Krol et al. (1988)
Biotechniques 6:958-976; and Stein et al. (1988) Cancer Res
48:2659-2668. With respect to antisense DNA,
oligodeoxyribonucleotides derived from the translation initiation
site, e.g., between the -10 and +10 regions of a Fra-1 protein
encoding nucleotide sequence, are preferred.
[0064] Antisense approaches involve the design of oligonucleotides
(either DNA or RNA) that are complementary to Fra-1 mRNA. The
antisense oligonucleotides will bind to Fra-1 mRNA transcripts and
prevent translation. Absolute complementarity, although preferred,
is not required. The ability to hybridize will depend on both the
degree of complementarity and the length of the antisense nucleic
acid. Generally, the longer the hybridizing nucleic acid, the more
base mismatches with an RNA it may contain and still form a stable
duplex or triplex. One skilled in the art can ascertain a tolerable
degree of mismatch by use of standard procedures to determine the
melting point of the hybridized complex. Oligonucleotides that are
complementary to the 5' end of the message, e.g., the 5'
untranslated sequence up to and including the AUG initiation codon,
should work most efficiently at inhibiting translation. However,
sequences complementary to the 3' untranslated sequences of mRNAs
have been shown to be effective at inhibiting translation of mRNAs
as well. (Wagner, R. (1994) Nature 372:333). Therefore,
oligonucleotides complementary to either the 5' or 3' untranslated,
non-coding regions of a Fra-1 gene could be used in an antisense
approach to inhibit translation of endogenous Fra-1 mRNA.
Oligonucleotides complementary to the 5' untranslated region of the
mRNA should preferably include the complement of the AUG start
codon. Although antisense oligonucleotides complementary to mRNA
coding regions are generally less efficient inhibitors of
translation, these could still be used in the invention. Whether
designed to hybridize to the 5', 3' or coding region of a Fra-1
mRNA, preferred antisense nucleic acids are less that about 100
(e.g., less than about 30, 25, 20, or 18) nucleotides in length.
Generally, in order to be effective, the antisense oligonucleotide
should be 18 or more nucleotides in length.
[0065] Specific antisense oligonucleotides can be tested for
effectiveness using in vitro studies to assess the ability of the
antisense oligonucleotide to inhibit gene expression. Preferably
such studies (1) utilize controls (e.g., a non-antisense
oligonucleotide of the same size as the antisense oligonucleotide)
to distinguish between antisense gene inhibition and nonspecific
biological effects of oligonucleotides, and (2) compare levels of
the target RNA or protein with that of an internal control RNA or
protein.
[0066] Antisense oligonucleotides of the invention may include at
least one modified base or sugar moiety. Exemplary modified bases
include 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,
5-(carboxyhydroxyethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluraci- l, dihydrouricil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-idimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopenten- yladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Exemplary modified sugar moieties include
arabinose, 2-fluoroarabinose, xylulose, and hexose. The antisense
oligonucleotides of the invention may in some embodiments include
at least one modified phosphate backbone such as a
phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a
phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl
phosphotriester, or a formacetal or analog thereof.
[0067] Antisense oligonucleotides within the invention might also
be an alpha-anomeric oligonucleotide. See, Gautier et al. (1987)
Nucl. Acids Res. 15:6625-6641. For example, the antisense
oligonucleotide can be a 2'-0-methylribonucleotide (Inoue et al.
(1987) Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA
analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).
Oligonucleotides of the invention may be synthesized by standard
methods known in the art, e.g by use of an automated DNA
synthesizer. Phosphorothioate oligonucleotides may be synthesized
by the method of Stein et al. (1988) Nucl. Acids Res. 16:3209).
Methylphosphonate oligonucleotides can be prepared by use of
controlled pore glass polymer supports (Sarin et al. (1988) Proc.
Natl. Acad. Sci. U.S.A. 85:7448-7451).
[0068] Methods of the invention also utilize techniques for
delivering one or more of the above-described nucleic acid
molecules into cells that express Fra-1. A number of methods have
been developed for delivering antisense DNA or RNA into cells. For
instance, antisense molecules can be introduced directly into a
cell by electroporation, liposome-mediated transfection,
CaCl-mediated transfection, viral vector infection, or using a gene
gun. Modified nucleic acid molecules designed to target the desired
cells (e.g., antisense oligonucleotides linked to peptides or
antibodies that specifically bind receptors or antigens expressed
on the target cell surface) can be used. To achieve high
intracellular concentrations of antisense oligonucleotides (as may
be required to suppress translation on endogenous mRNAs), a
preferred approach utilizes a recombinant DNA construct in which
the antisense oligonucleotide is placed under the control of a
strong promoter (e.g., the CMV promoter).
[0069] Ribozyme molecules designed to catalytically cleave Fra-1
mRNA transcripts can also be used to prevent translation of Fra-1
mRNAs and expression of Fra-1 proteins (See, e.g., Wright and
Kearney, Cancer Invest. 19:495, 2001; Lewin and Hauswirth, Trends
Mol. Med. 7:221, 2001; Sarver et al. (1990) Science 247:1222-1225
and U.S. Pat. No. 5,093,246). As one example, hammerhead ribozymes
that cleave mRNAs at locations dictated by flanking regions that
form complementary base pairs with the target mRNA might be used so
long as the target mRNA has the following common sequence:
5'-UG-3'. See, e.g., Haseloff and Gerlach (1988) Nature
334:585-591. To increase efficiency and minimize the intracellular
accumulation of non-functional mRNA transcripts, a ribozyme should
be engineered so that the cleavage recognition site is located near
the 5' end of the target Fra-1 mRNA. Ribozymes within the invention
can be delivered to a cell using a vector as described below.
[0070] Other methods can also be used to reduce Fra-1 gene
expression in a cell. For example, Fra-1 gene expression can be
reduced by inactivating or "knocking out" the Fra-1 gene or its
promoter using targeted homologous recombination. See, e.g, Kempin
et al., Nature 389: 802 (1997); Smithies et al. (1985) Nature
317:230-234; Thomas and Capecchi (1987) Cell 51:503-512; and
Thompson et al. (1989) Cell 5:313-321. For instance, a mutant,
non-functional Fra-1 gene variant (or a completely unrelated DNA
sequence) flanked by DNA homologous to the endogenous Fra-1 gene
(either the coding regions or regulatory regions of the Fra-1 gene)
can be used, with or without a selectable marker and/or a negative
selectable marker, to transfect cells that express Fra-1 protein in
vivo.
[0071] Fra-1 gene expression might also be reduced by targeting
deoxyribonucleotide sequences complementary to the regulatory
region of the Fra-1 gene (i.e., the Fra-1 promoter and/or
enhancers) to form triple helical structures that prevent
transcription of the Fra-1 gene in target cells. See generally,
Helene, C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C., et
al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992)
Bioassays 14(12):807-15. Nucleic acid molecules to be used in this
technique are preferably single stranded and composed of
deoxyribonucleotides. The base composition of these
oligonucleotides should be selected to promote triple helix
formation via Hoogsteen base pairing rules, which generally require
sizable stretches of either purines or pyrimidines to be present on
one strand of a duplex. Nucleotide sequences may be
pyrimidine-based, which will result in TAT and CGC triplets across
the three associated strands of the resulting triple helix. The
pyrimidine-rich molecules provide base complementarity to a
purine-rich region of a single strand of the duplex in a parallel
orientation to that strand. In addition, nucleic acid molecules may
be chosen that are purine-rich, for example, containing a stretch
of G residues. These molecules will form a triple helix with a DNA
duplex that is rich in GC pairs, in which the majority of the
purine residues are located on a single strand of the targeted
duplex, resulting in CGC triplets across the three strands in the
triplex. The potential sequences that can be targeted for triple
helix formation may be increased by creating a so called
"switchback" nucleic acid molecule. Switchback molecules are
synthesized in an alternating 5'-3', 3'-5' manner, such that they
base pair with first one strand of a duplex and then the other,
eliminating the necessity for a sizable stretch of either purines
or pyrimidines to be present on one strand of a duplex.
[0072] The antisense RNA and DNA, ribozyme, and triple helix
molecules that can be used with methods of the invention may be
prepared by any method known in the art for the synthesis of DNA
and RNA molecules. These include techniques for chemically
synthesizing oligodeoxyribonucleotides and oligoribonucleotides
well known in the art such as for example solid phase phosphoramide
chemical synthesis. RNA molecules may be generated by in vitro and
in vivo transcription of DNA sequences encoding the antisense RNA
molecule. Such DNA sequences may be incorporated into a wide
variety of vectors which incorporate suitable RNA polymerase
promoters. Alternatively, antisense cDNA constructs that synthesize
antisense RNA constitutively or inducibly, depending on the
promoter used, can be introduced stably into cell lines.
Inhibiting VEGF-D Expression
[0073] Methods that can be used to reduce VEGF-D expression include
modulating the expression and activity of Fra-1. The gene for
VEGF-D harbors an optimal AP-1 binding site in its promoter region
and is activated by Fra-1, an AP-1 transcription factor.
Accordingly, one may modulate VEGF-D expression by interfering with
the binding of the Fra-1 protein to the VEGF-D promoter.
[0074] More particularly, methods of the invention may involve
targeting the interaction of Fra-1 with its binding partners in an
effort to block activation of the VEGF-D promoter by Fra-1. Fra-1
cannot activate gene expression itself, as it requires
heterodimerization with Jun proteins to do so. c-Jun and JunB in
particular are preferable partners for Fra-1 and, in the process of
Fra-1 upregulation in response to Ras activation, c-Jun is
primarily utilized as the binding partner with Fra-1. Therefore,
nucleic acids which encode proteins that bind Fra-1 and preclude
binding of Fra-1 to c-Jun may be used to block activation of the
VEGF-D promoter by Fra-1. Methods of the invention may
alternatively utilize mimetics, e.g. peptide or non-peptide agents,
that are able to disrupt binding of a Fra-1 protein to other
proteins or molecules (e.g., c-Jun) with which the native Fra-1
protein interacts. Alternatively, antagonistic forms of the Fra-1
protein can be generated which are able to inhibit the function of
the naturally occurring form of the protein, such as by
competitively binding to another molecule that interacts with Fra-1
protein. In particular, a variant form of Fra-1 that is able to
heterodimerize with c-Jun yet is incapable of binding the VEGF-D
promoter is suitable for use in a method to inhibit VEGF-D gene
expression. Similarly, a variant form of c-Jun that is able to
heterodimerize with Fra-1 yet is incapable of binding the VEGF-D
promoter is suitable for use in a method to inhibit VEGF-D gene
expression. Fra-1 and c-Jun protein variants can be generated
through various techniques known in the art. For example, Fra-1 and
c-Jun protein variants can be made by mutagenesis, such as by
introducing an insertion, deletion or a discrete point
mutation(s).
Gene Therapy
[0075] Methods of the present invention include the delivery of
nucleic acids and proteins into a mammalian subject for inhibiting
angiogenesis or otherwise treating a cancer. Gene therapy can be
defined as the treatment of inherited or acquired diseases by the
introduction and expression of genetic information in cells.
Methods and compositions involving gene therapy vectors are
described herein. Such techniques are generally known in the art
and are described in methodology references such as Viral Vectors,
eds. Yakov Gluzman and Stephen H. Hughes, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1988; Retroviruses,
Cold Spring Harbor Laboratory Press, Plainview, N.Y., 2000; Gene
Therapy Protocols (Methods in Molecular Medicine), ed. Jeffrey R.
Morgan, Humana Press, Totawa, N.J., 2001; and Molecular Cloning: A
Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
[0076] Methods for inhibiting angiogenesis include anti-angiogenic
agents that may be administered to a mammalian subject, including a
human, by any suitable technique. Various techniques using viral
vectors for the introduction of nucleic acids encoding a Fra-1
variant, Fra-1 binding species, c-Jun binding species, and
antisense constructs into cells may be utilized in methods of the
invention. Viruses are naturally evolved vehicles which efficiently
deliver their genes into host cells and therefore are desirable
vector systems for the delivery of therapeutic genes. Preferred
viral vectors exhibit low toxicity to the host cell and produce
therapeutic quantities of anti-angiogenic compounds in a
tissue-specific manner. Viral vector methods and protocols are
reviewed in Kay et al. Nature Medicine 7:33-40, 2001.
[0077] Methods for use of recombinant Adenoviruses as gene therapy
vectors are discussed, for example, in W. C. Russell, Journal of
General Virology 81:2573-2604, 2000, and Bramson et al., Curr.
Opin. Biotechnol. 6:590-595, 1995. Adenovirus vectors have been
shown to be capable of highly efficient gene expression in target
cells and allow for a large coding capacity of heterologous DNA.
Heterologous DNA in this context may be defined as any nucleotide
sequence or gene which is not native to the Adenovirus. A preferred
form of recombinant Adenovirus is a "gutless", "high-capacity", or
"helper-dependent" Adenovirus vector which has all viral coding
sequences deleted, and contains the viral inverted terminal repeats
(ITRs), therapeutic gene (including Fra-1 binding species)
sequences (up to 28-32 kb) and the viral DNA packaging sequence.
Variants of such recombinant Adenovirus vectors such as vectors
containing tissue-specific (e.g., brain) enhancers and promoters
operably linked to a nucleic acid encoding a Fra-1 variant, Fra-1
binding species, c-Jun binding species, and antisense constructs
are also within the invention. More than one promoter can be
present in a vector. Accordingly, more than one heterologous gene
can be expressed by a vector. Further, the vector can include a
sequence which facilitates the localization of the Fra-1 variant,
Fra-1 binding species, and c-Jun binding species proteins and
antisense constructs to the nucleus of the cell, for example.
[0078] The viral vectors of the present invention can also include
Adeno-Associated Virus (AAV) vectors. AAV exhibits a high
transduction efficiency of target cells and can integrate into the
host genome in a site-specific manner. Methods for use of
recombinant AAV vectors are discussed, for example, in Tal, J., J.
Biomed. Sci. 7:279-291, 2000 and Monahan and Samulski, Gene Therapy
7:24-30, 2000. A preferred AAV vector comprises a pair of AAV
inverted terminal repeats which flank at least one cassette
containing a promoter which directs tissue (e.g., brain) or
cell-specific (e.g., astrocytoma) expression operably linked to a
nucleic acid encoding a Fra-1 variant, Fra-1 binding species, c-Jun
binding species, or antisense construct. The DNA sequence of the
AAV vector, including the ITRs, the promoter and the
anti-angiogenic agent may be integrated into the host genome.
[0079] Methods for use of Herpes Simplex Virus (HSV) vectors are
discussed, for example, in Cotter and Robertson, Curr. Opin. Mol.
Ther. 1:633-644, 1999. HSV vectors deleted of one or more immediate
early genes (IE) are non-cytotoxic, persist in a state similar to
latency in the host cell, and afford efficient host cell
transduction. Recombinant HSV vectors allow for approximately 30 kb
of coding capacity. A preferred HSV vector is engineered from HSV
type I, is deleted of the immediate early genes (IE) and contains a
tissue-specific (e.g., brain) promoter operably linked to a nucleic
acid encoding a Fra-1 variant, Fra-1 binding species, c-Jun binding
species, or antisense construct. HSV amplicon vectors may also be
used according to the invention. Typically, HSV amplicon vectors
are approximately 15 kb in length, possess a viral origin of
replication and packaging sequences. More than one promoter can be
present in a vector. Accordingly, more than one heterologous gene
can be expressed by a vector.
[0080] Viral vectors of the present invention may also include
replication-defective lentiviral vectors, including HIV. Methods
for use of lentiviral vectors are discussed, for example, in Vigna
and Naldini, J. Gene Med. 5:308-316, 2000 and Miyoshi et al., J.
Virol. 72:8150-8157, 1998. Lentiviral vectors are capable of
infecting both dividing and non-dividing cells and efficient
transduction of epithelial tissues of humans. HIV vectors have been
shown to efficiently infect hepatic cells. Lentiviral vectors
according to the invention may be derived from human and non-human
(including SIV) lentiviruses. A preferred lentiviral vector of the
present invention may include nucleic acid sequences required for
vector propagation in addition to a tissue-specific promoter (e.g.,
brain) operably linked to a nucleic acid encoding a variant Fra-1
protein, Fra-1 binding species, c-Jun binding species or antisense
construct. These sequences may include the viral LTRs, primer
binding site, polypurine tract, att sites and encapsidation site.
The lentiviral vector may be packaged into any suitable lentiviral
capsid. The substitution of one particle protein by one from a
different virus is referred to as "pseudotyping". The vector capsid
may contain viral envelope proteins from other viruses, including
Murine Leukemia Virus (MLV) or Vesicular Stomatitis Virus (VSV).
The use of the VSV G-protein yields a high vector titer and results
in greater stability of the vector virus particles. More than one
promoter can be present in a vector. Accordingly, more than one
heterologous gene can be expressed by a vector.
[0081] The invention also provides for use of retroviral vectors,
including Murine Leukemia Virus-based vectors. Methods for use of
retrovirus-based vectors are discussed, for example, in Hu and
Pathak, Pharmacol. Rev. 52:493-511, 2000 and Fong et al., Crit.
Rev. Ther. Drug Carrier Syst. 17:1-60, 2000. Retroviral vectors
according to the invention may contain up to 8 kb of heterologous
(therapeutic) DNA, in place of the viral genes. Heterologous may be
defined in this context as any nucleotide sequence or gene which is
not native to the retrovirus. The heterologous DNA may include a
tissue-specific promoter, a nucleic acid encoding a variant Fra-1
protein, Fra-1 binding species, c-Jun binding species or antisense
construct and may encode a ligand to a brain cell-specific
receptor. The retroviral particle may be pseudotyped, and may
contain a viral envelope glycoprotein from another virus, in place
of the native retroviral glycoprotein. The retroviral vector of the
present invention may integrate into the genome of the host cell.
More than one promoter can be present in a vector. Accordingly,
more than one heterologous gene can be expressed by a vector
[0082] Other viral vectors within the invention are Alphaviruses,
including Semliki Forest Virus (SFV) and Sindbis Virus (SIN).
Methods for use of Alphaviruses are described, for example, in
Lundstrom, K., Intervirology 43:247-257, 2000 and Perri et al.,
Journal of Virology 74:9802-9807, 2000. Alphavirus vectors
typically are constructed in a format known as a replicon. Such
replicons may contain Alphavirus genetic elements required for RNA
replication, as well as expression of a nucleic acid encoding a
variant Fra-1 protein, Fra-1 binding species, c-Jun binding species
or antisense construct. Heterologous may be defined in this context
as any nucleotide sequence or gene which is not native to the
Alphavirus. Within the Alphivirus replicon, the nucleic acid
encoding a variant Fra-1 protein, Fra-1 binding species, c-Jun
binding species or antisense construct may be operably linked to a
tissue-specific (e.g., brain) promoter or enhancer. Recombinant,
replication-defective Alphavirus vectors are capable of high-level
heterologous (therapeutic) gene expression, and can infect a wide
host cell range. Alphavirus replicons according to the invention
may be targeted to specific cell types (e.g., astrocytes) by
displaying on their virion surface a functional heterologous ligand
or binding domain that would allow selective binding to target
cells expressing the cognate binding partner. Alphavirus replicons
according to the invention may establish latency, and therefore
long-term expression of a nucleic acid encoding a variant Fra-1
protein, Fra-1 binding species, c-Jun binding species or antisense
construct in the host cell. The replicons may also exhibit
transient expression of a nucleic acid encoding a variant Fra-1
protein, Fra-1 binding species, c-Jun binding species or antisense
construct in the host cell. A preferred Alphavirus vector or
replicon of the invention is noncytopathic. More than one promoter
can be present in a vector. Accordingly, more than one heterologous
gene can be expressed by a vector.
[0083] To combine advantageous properties of two viral vector
systems, hybrid viral vectors may be used to deliver an
anti-angiogenic agent to a target tissue (e.g., brain). Standard
techniques for the construction of hybrid vectors are well-known to
those skilled in the art. Such techniques can be found, for
example, in Sambrook, et al., In Molecular Cloning: A laboratory
manual. Cold Spring Harbor, N.Y. or any number of laboratory
manuals that discuss recombinant DNA technology. Double-stranded
AAV genomes in adenoviral capsids containing a combination of AAV
and Adenoviral ITRs may be used to transduce cells. In another
variation, an AAV vector may be placed into a "gutless",
"helper-dependent" or "high-capacity" Adenoviral vector.
Adenovirus/AAV hybrid vectors are discussed in Lieber et al., J.
Virol. 73:9314-9324, 1999. Retroviral/Adenovirus hybrid vectors are
discussed in Zheng et al., Nature Biotechnol. 18:176-186, 2000.
Retroviral genomes contained within an Adenovirus may integrate
within the host cell genome and effect stable expression of a
nucleic acid encoding a variant Fra-1 protein, Fra-1 binding
species, c-Jun binding species or antisense construct. More than
one promoter can be present in a vector. Accordingly, more than one
heterologous gene can be expressed by a vector.
[0084] In accordance with the present invention, other nucleotide
sequence elements which facilitate expression of the
anti-antiangiogenic agent and cloning of the vector are further
contemplated. The presence of enhancers upstream of the promoter or
terminators downstream of the coding region, for example, can
facilitate expression. In the vectors of the present invention, the
presence of elements which enhance brain-specific expression of a
nucleic acid encoding a Fra-1 protein, Fra-1 binding species, c-Jun
binding species or antisense construct may be useful for gene
therapy.
[0085] Several non-viral methods for introducing an anti-angiogenic
agent into host cells are also within the scope of the invention.
For a review of non-viral methods, see Nishikawa and Huang, Human
Gene Ther. 12:861-870, 2001. Various techniques employing plasmid
DNA for the introduction of a nucleic acid encoding a variant Fra-1
protein, Fra-1 binding species, c-Jun binding species or antisense
construct into cells may be utilized in methods of the invention.
Such techniques are generally known in the art and are described in
references such as Ilan, Y., Curr. Opin. Mol. Ther. 1:116-120,
1999, Wolff, J. A., Neuromuscular Disord. 7:314-318, 1997 and
Arztl, Z., Fortbild Qualitatssich 92:681-683, 1998.
[0086] Methods involving physical techniques for the introduction
of an anti-angiogenic agent into a host cell can be adapted for use
in the present invention. The particle bombardment method of gene
transfer involves an Accell device (gene gun) to accelerate
DNA-coated microscopic gold particles into target tissue, including
the brain. Particle bombardment methods are described in Yang et
al., Mol. Med. Today 2:476-481 1996 and Davidson et al., Rev. Wound
Repair Regen. 6:452-459, 2000. Cell electropermeabilization (also
termed cell electroporation) may be employed for delivery of a
nucleic acid encoding a variant Fra-1 protein, Fra-1 binding
species, c-Jun binding species or antisense construct into cells of
tissues. This technique is discussed in Preat, V., Ann. Pharm. Fr.
59:239-244 2001 and involves the application of pulsed electric
fields to cells to enhance cell permeability, resulting in
exogenous polynucleotide transit across the cytoplasmic
membrane.
[0087] Synthetic gene transfer molecules according to the invention
can be designed to form multimolecular aggregates with plasmid DNA
(harboring a nucleic acid encoding a variant Fra-1 protein, Fra-1
binding species, c-Jun binding species or antisense construct
operably linked to a brain-specific promoter) and to bind the
resulting particles to the target cell (e.g., astrocytes) surface
in such a way as to trigger endocytosis and endosomal membrane
disruption. Polymeric DNA-binding cations (including polylysine,
protamine, and cationized albumin) can be linked to
astrocyte-specific targeting ligands and trigger receptor-mediated
endocytosis into astrocytes. Methods involving polymeric
DNA-binding cations are reviewed in Guy et al., Mol. Biotechnol.
3:237-248, 1995 and Garnett, M. C., Crit. Rev. Ther. Drug Carrier
Syst. 16:147-207, 1999. Cationic amphiphiles, including
lipopolyamines and cationic lipids, may provide
receptor-independent transfer of a nucleic acid encoding a variant
Fra-1 protein, Fra-1 binding species, c-Jun binding species or
antisense construct into target cells (e.g., astrocytes). Preformed
cationic liposomes or cationic lipids may be mixed with plasmid DNA
to generate cell transfecting complexes. Methods involving cationic
lipid formulations are reviewed in Felgner et al., Ann. N.Y. Acad.
Sci. 772:126-139, 1995 and Lasic and Templeton, Adv. Drug Delivery
Rev. 20:221-266, 1996. Suitable methods can also include use of
cationic liposomes as agents for introducing DNA or protein into
cells. For therapeutic gene delivery, DNA may also be coupled to an
amphipathic cationic peptide (Fominaya et al., J. Gene Med.
2:455-464, 2000).
[0088] Methods that involve both viral and non-viral based
components may be used according to the invention. An Epstein Barr
Virus (EBV) based plasmid for therapeutic gene delivery is
described in Cui et al., Gene Therapy 8:1508-1513, 2001. A method
involving a DNA/ligand/polycationic adjunct coupled to an
Adenovirus is described in Curiel, D. T., Nat. Immun. 13:141-164,
1994. More than one promoter can be present in a vector.
Accordingly, more than one heterologous gene can be expressed by a
vector.
[0089] Other techniques according to the invention may be based on
the use of brain-specific ligands. Synthetic peptides or
polypeptides may be used as ligands in targeted delivery of DNA and
proteins to brain-specific receptors. Complexes of protein and
ligand or plasmid DNA and ligand mediate protein and DNA transfer
into brain cells.
[0090] Methods involving ultrasound contrast agent delivery
vehicles may be used in the invention. Such methods are discussed
in Newman et al., Echocardiography 18:339-347, 2001 and Lewin et
al. Invest. Radiol. 36:9-14, 2001. Gene-bearing microbubbles, when
exposed to ultrasound, cavitate and locally release a therapeutic
agent. Attachment of a brain cell-targeting moiety to the contrast
agent vehicle may result in site-specific (e.g., brain) expression
of a nucleic acid encoding a variant Fra-1 protein, Fra-1 binding
species, c-Jun binding species or antisense construct.
[0091] Methods which are well known to those skilled in the art can
be used to construct a natural or synthetic matrix that provides
support for the delivered agent (e.g., an anti-angiogenic agent)
prior to delivery. See, for example, the techniques described in
Murphy and Mooney, J. Period Res., 34:413-9, 1999 and Vercruysse
and Prestwich, Crit. Rev. Ther. Drug Carrier Syst., 15:513-55,
1998. The particular type of matrix used can be any suitable matrix
for use in the invention. For implantation into an animal subject,
preferred matrix will have all the features commonly associated
with being "biocompatible", in that they do not produce an adverse,
or allergic reaction when administered to the recipient host.
Matrices suitable for use in the invention may be formed from both
natural or synthetic materials and may be designed to allow for
sustained release of the therapeutic agent and growth factors over
prolonged periods of time. Thus, appropriate matrices will both
provide anti-angiogenic factors and also act as an in situ
scaffolding for the delivered agent (e.g., a nucleic acid encoding
a variant Fra-1 protein, Fra-1 binding species, c-Jun binding
species or antisense construct). Preferred matrices are those that
are biodegradable as these are capable of being reabsorbed.
[0092] Delivery of an anti-angiogenic agent, according to the
invention, may involve methods of DNA microencapsulation.
Microparticles, also known as microcapsules and microspheres, may
be used as gene delivery vehicles. They may be delivered in
operable form noninvasively to epithelial surfaces for gene
therapy. The genes within the microparticles can pass across
epithelial barriers and travel to remote sites, via systemic
circulation. Microencapsulated gene delivery vehicles may be
constructed from low viscosity polymer solutions that are forced to
phase invert into fragmented spherical polymer particles when added
to appropriate nonsolvents. Methods involving microparticles are
discussed in Hsu et al., J. Drug Target 7:313-323, 1999 and Capan
et al., Pharm. Res. 16:509-513, 1999.
[0093] Methods involving microencapsulated recombinant cells may be
used in the invention. Such an approach may be used in either in
vivo or ex vivo techniques. Cells that contain an expression vector
coding for a nucleic acid encoding a variant Fra-1 protein, Fra-1
binding species, c-Jun binding species or antisense construct or
that have been engineered to stably express a nucleic acid encoding
a variant Fra-1 protein, Fra-1 binding species, c-Jun binding
species or antisense construct may be encapsulated in microcapsules
that provide protection from immune mediators and allow appropriate
release of the anti-angiogenic agent. Preferred microencapsulation
particles, also referred to as encapsulation devices, consist of
biocompatible and biodegradable components. Techniques involving
microencapsulated cells are discussed in Ross et al. Hum. Gen.
Ther. 11:2117-2127, 2000 and Fong et al., Crit. Rev. Ther. Drug
Carrier Syst. 17:1-60, 2000.
[0094] Protein transduction offers an alternative to gene therapy
for the delivery of therapeutic proteins into target cells, and
methods of protein transduction are within the scope of the
invention. Protein transduction is the internalization of proteins
into a host cell, from the external environment. The
internalization process relies on a protein or peptide which is
able to penetrate the cell membrane. The transducing property of
such a protein or peptide can be conferred upon proteins (Fra-1
variant, Fra-1 binding species, and a c-Jun binding species, for
example) which are expressed as fusion proteins with them. Commonly
used protein transduction vehicles include the antennapedia
peptide, the HIV TAT protein transduction domain and the herpes
simplex virus VP22 protein. Such vehicles are reviewed in Ford et
al., Gene Ther. 8:1-4, 2001.
Method for Identifying a Test Compound
That Modulates Fra-1 Gene Expression
[0095] The invention provides for a method of identifying a test
compound that modulates expression of a Fra-1 gene in a brain
cancer cell. One such method involves providing a cell that
expresses Fra-1 and at least one test compound, contacting the cell
with the test compound, and detecting whether or not the test
compound modulates Fra-1 expression. Those compounds resulting
specifically in altered levels (increased or decreased levels) of
Fra-1 protein are those that specifically modulate Fra-1
expression. For example, a library of molecules can be screened by
providing brain cancer cells expressing Fra-1 and contacting the
cells with the library and examining the cells for changes in Fra-1
expression. Changes in Fra-1 expression may be assessed by
analyzing changes in Fra-1 marker (e.g. Fra-1 protein and Fra-1
mRNA) levels.
Disruption of Fra-1/c-JUN-VEGF-D Promoter Interactions
[0096] Nucleic acids encoding binding mutants as well as binding
mutant proteins may be used in methods of the invention to
interfere with binding of Fra-1 and/or c-Jun to the VEGF-D gene
promoter. Such molecules include a variant Fra-1 protein that binds
to native c-Jun yet lacks the ability to bind the VEGF-D promoter.
An example of such a variant is a dominant negative mutant of Fra-1
which dimerizes with native c-Jun and blocks binding of the dimer
to a VEGF-D promoter. Similarly, an example of another variant is a
dominant negative mutant of c-Jun which dimerizes with native Fra-1
and blocks binding of the dimer to a VEGF-D promoter.
Alternatively, nucleic acids themselves may be used to disrupt the
binding of these transcription factors to the VEGF-D promoter. For
example, over-expression of a high copy plasmid harboring an excess
of AP-1 binding sites (i.e. VEGF-D promoter binding sites) would
bind Fra-1 and sequester Fra-1 from the native VEGF-D promoter. The
mutagenic techniques described herein can be used to map which
determinants of Fra-1 and c-Jun proteins participate in the
intermolecular interactions involved in, for example, binding of
Fra-1 or c-Jun to a VEGF-D promoter.
[0097] Fra-1 and c-Jun protein variants that do not bind a VEGF-D
promoter can be generated through various techniques known in the
art. For example, Fra-1 and c-Jun protein variants can be made by
mutagenesis, such as by introducing discrete point mutation(s), by
insertion or deletion. Whether a change in the amino acid sequence
of a peptide results in a Fra-1 or c-Jun protein variant lacking
one or more functional activities of a native Fra-1 or c-Jun
protein can be readily determined by testing the variant for a
native Fra-1 or c-Jun protein functional activity. For example, a
brain tissue sample containing a VEGF-D promoter receptor can be
contacted with a Fra-1 or c-Jun protein variant that lacks the
ability to bind the promoter. The brain tissue sample can then be
analyzed for VEGF-D gene expression as well as angiogenesis.
Inhibiting Angiogenesis
[0098] The invention provides a method for inhibiting angiogenesis
associated with a brain cancer in a subject by providing an agent
that interferes with Fra-1 binding to a VEGF-D gene promoter and
administering the agent to the central nervous system of the
subject. The agent would be administered in an amount effective to
inhibit blood vessel development associated with cancer. For
example, VEGF-D expression in a brain cell may be inhibited by
introducing into the cell an agent that interferes with activation
of the VEGF-D gene by Fra-1. Such an agent can be an
oligonucleotide
[0099] (e.g., antisense oligonucleotide) that hybridizes to a
polynucleotide that encodes a Fra-1 protein. In another embodiment,
the agent may be a protein that binds Fra-1 and precludes the
interaction of Fra-1 with its binding partner c-Jun.
Administration of Compositions
[0100] The compositions described above may be administered to
animals including human beings in any suitable formulation. For
example, anti-angiogenic molecules may be formulated in
pharmaceutically acceptable carriers or diluents such as
physiological saline or a buffered salt solution. Suitable carriers
and diluents can be selected on the basis of mode and route of
administration and standard pharmaceutical practice. A description
of exemplary pharmaceutically acceptable carriers and diluents, as
well as pharmaceutical formulations, can be found in Remington's
Pharmaceutical Sciences, a standard text in this field, and in
USP/NF. Other substances may be added to the compositions to
stabilize and/or preserve the compositions.
[0101] The compositions of the invention may be administered to
animals by any conventional technique. The compositions may be
administered directly to a target site by, for example, surgical
delivery to an internal or external target site, or by catheter to
a site accessible by a blood vessel. Other methods of delivery,
e.g., liposomal delivery or diffusion from a device impregnated
with the composition, are known in the art. The compositions may be
administered in a single bolus, multiple injections, or by
continuous infusion (e.g., intravenously). For parenteral
administration, the compositions are preferably formulated in a
sterilized pyrogen-free form.
[0102] Systemic (i.v.) with local interstitial drug delivery may be
used according to the invention. The concept of convection enhanced
delivery is becoming more attractive as an effective route of drug
delivery into the brain. Laske et al., Nature Medicine 3, 1362-1368
(1997). Consequently, local delivery is the preferred approach to
be evaluated clinically, since it may achieve high concentrations
directly within the tumor mass and its vicinity.
[0103] Generally, compositions used in methods of the invention are
introduced into a tumor cell using in vivo transduction techniques.
Particularly, for in vivo delivery, the compositions will be
formulated into pharmaceutical compositions and generally
administered by direct injection into a tumor mass, injected
intravenously into blood veins feeding the tumor mass, or
administered into a tumor bed subsequent to tumor resection.
[0104] The compositions used in the invention may be precisely
delivered into tumor sites, e.g., into gliomas or other
intracranial tumors, by using stereotactic microinjection
techniques. For example, the mammalian subject to be treated can be
placed within a stereotactic frame base that is MRI-compatible and
then imaged using high resolution MRI to determine the
three-dimensional positioning of the particular tumor being
treated. According to this technique, the MRI images are then
transferred to a computer having the appropriate stereotactic
software, and a number of images are used to determine a target
site and trajectory for anti-angiogenic composition microinjection.
Using such software, the trajectory is translated into
three-dimensional coordinates appropriate for the stereotactic
frame. For intracranial delivery, the skull will be exposed, burr
holes will be drilled above the entry site, and the stereotactic
apparatus positioned with the needle implanted at a predetermined
depth. Tumor resection operations may be carried out prior to
positioning of the stereotactic apparatus, if desired. A
pharmaceutical composition containing an anti-angiogenic agent
according to the invention can then be microinjected at the
selected target site(s).
Effective Doses
[0105] The compositions described above are preferably administered
to a mammal in an effective amount, that is, an amount capable of
producing a desirable result in a treated subject (e.g., inhibiting
angiogenesis and treating malignant tumors in the subject). Such a
therapeutically effective amount can be determined as described
below.
[0106] Toxicity and therapeutic efficacy of the compositions
utilized in methods of the invention can be determined by standard
pharmaceutical procedures, using either cells in culture or
experimental animals to determine the LD.sub.50 (the dose lethal to
50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index and it can be
expressed as the ratio LD.sub.50/ED.sub.50. Those compositions that
exhibit large therapeutic indices are preferred. While those that
exhibit toxic side effects may be used, care should be taken to
design a delivery system that minimizes the potential damage of
such side effects. The dosage of preferred compositions lies
preferably within a range that includes an ED.sub.50 with little or
no toxicity. The dosage may vary within this range depending upon
the dosage form employed and the route of administration
utilized.
[0107] As is well known in the medical and veterinary arts, dosage
for any one animal depends on many factors, including the subject's
size, body surface area, age, the particular composition to be
administered, time and route of administration, general health, and
other drugs being administered concurrently. It is expected that an
appropriate dosage for intratumoral administration of the
compositions would be in the range of about 0.001 to 100 mg/kg body
weight.
EXAMPLES
[0108] The present invention is further illustrated by the
following specific examples. The examples are provided for
illustration only and are not to be construed as limiting the scope
or content of the invention in any way.
Example 1
Materials and Methods
[0109] Cell lines and tissues: Glioblastoma multiforme cells lines
A-172 MG, U-251 MG, DBTRG-50 MG, U-87 MG, U-373 MG, human GBM
explant cells, G48a, CSML0 and CSML100 mouse breast carcinoma cells
were grown in appropriate media. The CSML0 and CSML100 mouse breast
cancer cells and GBM A172 MG glioblastoma cells were grown in
Dulbecco's Modified Eagle's Medium (D-MEM) with 10% Fetal Calf
Serum (FCS) (Life Technologies, Rockville, Md.). U-251 MG cells
were grown in D-MEM, 10% FCS, 0.1 mM MEM Non-Essential Amino Acids
(NEAA) (Life Technologies), and 50 .mu.g/ml Gentamicin Sulfate. GBM
cell lines U-87 MG and U-373 MG were grown in Earle's Minimum
Essential Medium (MEM), 10% FCS, 0.1 mM NEAA, 2 mM Glutamine (Life
Technologies), and 100 .mu.g/ml Sodium Pyruvate. GMB cell line
DBTRG-50 MG and human explant cells were grown in RPMI-1640 (Life
Technologies) 10% FCS, 100 .mu.g/ml Sodium Pyruvate, 100 .mu.g/ml
L-Cystine (Life Technologies), 20 .mu.g/ml L-Proline (Sigma),
1.times.HT Supplement, consisting of 0.1 .mu.M Sodium Hypoxanthine
and 0.016 .mu.M Thymidine, 5 units/ml Pennicilin G and 5 units/ml
Streptomycin sulfate (Penn/Strep) (Life Technologies). Normal Human
Astrocytes (NHA) were grown in Astrocyte Growth Medium
BulletKit.TM. (BioWhittaker). Normal HUV-EC-C were grown in F-12
Kaighn's medium (Life Technologies) with 10% FCS, 100 .mu.g/ml
Heparin (Sigma) and 30 .mu.g/ml Endothelial Cell Growth Supplement
(ECGS) (Sigma).
[0110] A retroviral vector was used to generate plasmid pMvFra-1.
To produce replication-defective retroviruses, the GP+E packaging
cell line was employed, which was maintained in appropriate media.
Successfully transfected GP+E cells were selected in the presence
of 800 .mu.g/ml G418. Supernatants of virus-producing cell lines
were used to infect CSML0 cells. Infected cells were selected in
the presence of 400 .mu.g/ml G418.
[0111] GBM tumors and non-malignant brain tissue, the latter
obtained usually from the therapeutic resections for the treatment
of epilepsy, were obtained from the operating room and snap frozen
immediately, as described previously. Debinski et al. (1999) Clin.
Cancer Res. 5: 985-990. Ten-micron sections of GBM were
thaw-mounted onto chrom-alum slides. Slides were stored at
-80.degree. C. until assayed. Sections were allowed to thaw and
subsequently fixed for 10 min in acetone at -20.degree. C.
[0112] Immunostaining: GBM cells lines, human explant cells (G48a),
Human Umbilical Vein Endothelial Cells (HUV-EC-C) from ATCC
(Rockville, Md.), and normal human astrocytes (NHA) from
BioWhittaker (Walkersville, Md.) were grown overnight on sterile
glass slides in the appropriate media. Slides were washed twice in
PBS and fixed for 2 min in acetone at -80.degree. C. Slides were
washed twice in PBS and either used immediately or air-dried and
stored at -80.degree. C. until assayed. In stimulation experiments,
10.sup.4 SNB-19 GBM cells were plated on glass chamber slides and
allowed to attach overnight. The cells were washed with PBS and
serum-free media was applied. After 24 hr epidermal growth factor
(EGF) or leukemia inhibitory factor (LIF) were added to cells at 5
and 20 ng/ml, respectively. The cells were processed for
immunocytochemistry after 24 hr of stimulation period.
[0113] Mouse monoclonal anti-VEGF-D (VD1) antibody was used. See,
Achen et al. (2000) Eur. J. Biochem. 267: 2505-2515. It was
employed at a final dilution of 1:500 (7.5 .mu.g/ml). Other primary
antibodies including rabbit polyclonal Fra-1 (1:100), c-Fos
(1:100), c-Jun (1:150), and mouse monoclonal JunB (1:75) were
purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.); and
mouse monoclonal Factor VIII (1:150) and rabbit GFAP (1:500) were
purchased from DAKO Chemical (Carpinteria, Calif.).
[0114] Slides were washed in two changes of PBS and blocked for 30
min with 10% (v/v) normal goat serum (NGS) in PBS at room
temperature. Primary antibody was diluted in 1.5% NGS/PBS and
incubated at room temperature for either 1 hr (VEGF-D, Factor VIII,
and GFAP) or 2 hr (Fra-1, JunB, c-Fos, and c-Jun). Slides were
washed in three changes of PBS for 10 min each. Secondary antibody,
goat anti-rabbit Rhodamine (1:150), Jackson ImmunoResearch
Laboratories, Inc. (West Grove, Pa.) or sheep anti-mouse Cy3
(1:250), Sigma (St. Louis, Mo.) was diluted in 1.5% NGS/PBS and
incubated in the dark at room temperature for 45 min. For
double-labeling experiments, the secondary antibodies were goat
anti-mouse Oregon Green.TM. (1:200) (Molecular Probes, Oregon) and
goat anti-rabbit Rhodamine (1:150). Slides were washed in 3 changes
of PBS for 10 min each and mounted with Gel-Mount, Biomeda Corp.
(Foster City, Calif.). Some slides were counterstained with Hoechst
No. 33258 Nuclear Counterstain (DAPI).
[0115] Photomicrographs were taken at 40.times. magnification in
all cases with a Hamamatsu C2400 digital camera. Background was
normalized to the samples without primary antibody. Each set of
images was taken exactly at the same exposure settings. Images were
processed with Paint Shop Pro V 6.0 (Jasc software Inc., Eden
Prairie, Minn.).
[0116] Western Blots: Cell lysates were prepared from sub-confluent
cultures. Cells were washed twice in PBS and lysed in RIPA buffer
(PBS, 1% Igepal CA-630; ICN Biomedicals, Inc. Costa Mesa, Calif.),
0.5% sodium deoxycholate (Fisher Scientific, Fair lawn, N.J.), 0.1%
SDS containing Mammalian Protease Inhibitor Cocktail (Sigma). GBM
and non-malignant brain tumor samples were minced into small pieces
while frozen and thawed in RIPA buffer with Mammalian Protease
Inhibitor Cocktail. Lysates were passed through a 21-gauge needle
to shear the DNA. 1 mM PMSF (Sigma) was added and the lysates were
incubated on ice for 30-60 min. Non-solubilized debris was pelleted
at 10,000.times.g for 10 min. The supernatant was collected,
aliquoted, and stored at -80.degree. C. until use. Normal human
brain lysates were also purchased from Chemicon International, Inc.
(Temecula, Calif.) and Clontech.
[0117] Lysates were run on either 12% or 15% SDS-PAGE. Proteins
were transferred to PVDF membrane (Pierce, Rockford, Ill.) and
blocked for 1 hr with 5% blotto (5% dry milk, PBS, 0.05% Tween-20).
Membranes were incubated with primary antibody diluted in blotto
for 40 min at room temperature while shaking. Antibodies included:
anti-mouse VEGF-D antibody (40% cross-reactivity with human VEGF-D;
1:500) from R&D Systems, and Fra-1 (1:100) from Santa Cruz
Research Antibodies. Following three five-minute washes in
PBS/0.05% Tween-20, membranes were incubated in secondary antibody
conjugated with horseradish peroxidase (goat anti-mouse IgG or goat
anti-rabbit IgG) at a dilution of 1:10,000 or 1:15,000 in 5% blotto
for 40 min at room temperature while shaking. Membranes were washed
in several changes of PBS and detection was performed using the
SuperSignal West Pico Chemiluminescent Substrate (Pierce).
Membranes were exposed to autoradiographic film X-OMAT AR for up to
5 min. Films were scanned in a transparency scanner at a pixel size
of 88.times.88 micron (Molecular Dynamics, Sunnyvale, Calif.). The
images were compiled in Paint Shop Pro V 6.0.
[0118] cDNA arrays: Atlas Oncogene/Tumor Suppressor Arrays were
purchased from Clontech and 1 .mu.g of poly(A)+RNA was labeled with
[.alpha.-.sup.33P]dATP according to the manufacturer. Membranes
were pre-hybridized overnight at 68.degree. C. in ExpressHyb
(Clontech) containing 0.1 mg/ml sheared salmon sperm DNA. Labeled
cDNA probe was denatured and added to the pre-hybridization
solution and the membranes were hybridized overnight at 68.degree.
C. Membranes were then washed twice in 2.times.SSC/1% SDS for 20
min followed by two washes in 0.1% SSC/0.5% SDS at 68.degree. C.
The membranes were exposed to autoradiographic film for up to 10
days at -70.degree. C. The arrays contain cDNA specific fragments
for oncogenes, such as c-fos, junB, and c-myc. Housekeeping genes
included ubiquitin, liver glyceraldehyde 3-phosphate dehydrogenase
(GAPDH), and phospholipase. RNA used for the cDNA micro-array
assays was isolated from sub-confluent cultures of GBM cells using
the acid-guanidium isothiocyanate-phenol-chloroform method.
Chomczynski P, and Sacchi N (1987) Analyt. Biochem. 162: 156-159.
Poly(A)+RNA was further isolated using the Oligotex mRNA Kit
(Qiagen Inc, Valencia, Calif.). Normal Human Brain Poly(A)+RNA was
purchased from Clontech (Clontech Laboratories, Inc., Palo Alto,
Calif.).
[0119] Karyotyping: The karyotypes of HGA cells analyzed in this
study were performed in a blinded fashion by clinical
cytogeneticists at the Cancer Genetics Laboratory, Genetics &
IVF Institute, Fairfax, Va.
Example 2
Fos Transcription Factors in GBM Cells
[0120] VEGF-D has been reported as a c-fos inducible mitogenic and
morphogenic factor, and named accordingly a c-fos-induced growth
factor (FIGF). It was thus imperative to explore the c-fos oncogene
protein expression in GBM, since it was the foremost suspect
responsible for high and ubiquitous over-expression of VEGF-D.
Contrary to what one would expect, the levels of c-Fos in several
GBM cell lines were found to be low. Specific nuclear
immunoreactivity for c-Fos was seen mainly in some of the DBTRG-50
MG cells. Others found similarly low levels of c-fos gene
expression in brain tumor cells. Thus, different factors than c-Fos
may be involved in sustained VEGF-D up-regulation in GBM cells.
[0121] The gene for VEGF-D has an optimal AP-1 binding site in its
promoter region. Considering the lack of correlation between the
levels of c-Fos and VEGF-D in GBM cells, the possibility that other
AP-1 transcription factors, to which c-Fos belongs, play roles in
VEGF-D up-regulation was explored. Experiments with oncogene/tumor
suppressor gene-containing cDNA microarrays revealed that the
expression of the fos-related antigen-1 gene (Fra-1) is higher in
GBM cells, such as G48a, when compared with normal brain tissue,
while the expression of c-fos was usually undetectable. The same
phenomenon was observed in DBTRG-50 MG and U-87 MG GBM cell lines.
Therefore, immunofluorescence using anti-Fra-1 antibody was
performed in GBM cells. It was discovered that Fra-1 is highly
expressed in all GBM cell lines studied, such as G48a, U-87 MG,
U-251 MG, and DBTRG-50 MG. However, the pattern of staining for
Fra-1 was distinctly different from the rather diffuse cytoplasmic
staining seen for VEGF-D. Anti-Fra-1 immunopositivity was localized
to the nuclei of the cells, which would be expected for this
transcription factor. HUV-EC-C demonstrated limited nuclear
immunofluorescence for Fra-1. Western blot analysis was performed
on GBM cell lysates and tumor samples and revealed a protein band
of .about.42 kDa, which corresponds to the size of human Fra-1.
[0122] Fra-1 cannot activate gene expression itself, since it
requires heterodimerization with Jun proteins to do so. c-Jun and
JunB in particular are preferable partners for Fra-1 and, in the
process of Fra-1 up-regulation in response to Ras activation, c-Jun
was primarily utilized as the binding partner with Fra-1. Based on
cDNA microarray analyses, c-jun, and much less junB, was found
expressed in astrocytoma cells. In follow-up immunohistochemical
studies, the staining for c-Jun was readily detected and localized
to the nuclei of GBM cells, similar to the Fra-1 staining. JunB was
detected by immunohistochemistry although at lower intensities than
c-Jun. Interestingly, c-jun was the only gene expressed in normal
brain tissue among AP-1 factors.
Example 3
Fra-1 Induces Expression of VEGF-D
[0123] Studies have focused on Fra-1 and its role in
transcriptional activation of other factors which are likely
suspects involved in cancer progression/maintenance. For example,
Fra-1 has been previously identified as the primary AP-1 factor
involved in the development of a more invasive, highly progressive
carcinoma phenotype of breast cancer. Immunoblot analysis for
VEGF-D was therefore performed in cell lysates of CSML0 (low Fra-1)
and CSML100 (high Fra-1) mouse breast cancer cells. Elevated levels
of VEGF-D were observed in CSML100 when compared with CSMLO cells.
Furthermore, mock-transfected and Fra-1-transfected CSMLO cells
were utilized. The mock-transfected CSML0 cells did not express
Fra-1, as expected, and showed no VEGF-D immunoreactivity, whereas
the Fra-1 transgene evoked VEGF-D expression in transfected CSML0
cells. The size of the detected band was .about.33 kDa, which
corresponds to a form of murine VEGF-D, consisting most probably of
the N-terminal pro-peptide and the VEGF homology domain, that is
found in VEGF-D-producing organs, such as heart and in
VEGF-D-producing cells, such as fibroblasts. Thus, Fra-1 expression
converts cells to VEGF-D producers.
Other Embodiments
[0124] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
* * * * *